Methods and reagents for vaccination which generate a CD8 T cell immune response

ABSTRACT

New methods and reagents for vaccination are described which generate a CD8 T cell immune response against malarial and other antigens such as viral and tumour antigens. Novel vaccination regimes are described which employ a priming composition and a boosting composition, the boosting composition comprising a non-replicating or replication-impaired pox virus vector carrying at least one CD8 T cell epitope which is also present in the priming composition.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/833,745, filed Apr. 28, 2004, which is a continuation of U.S.application Ser. No. 10/686,943 (currently pending), filed Oct. 16,2003, which is a continuation of U.S. application Ser. No. 09/454,204,filed Dec. 9, 1999 (which issued as U.S. Pat. No. 6,663,871 B1), whichis a continuation of International Application No. PCT/GB98/01681, whichdesignated the United States and was filed Jun. 9, 1998, published inEnglish, and which claims priority under 35 U.S.C. §119 or 365 to GreatBritain Application No. GB9711957.2 filed Jun. 9, 1997.

The entire teachings of the above applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

A general problem in vaccinology has been an inability to generate highlevels of CD8 T cells by immunization. This has impeded the developmentof vaccines against several diseases including malaria.

Plasmodium falciparum malaria causes hundreds of millions of malariainfections each year and is responsible for 1-2 million deaths annually.The development of an effective vaccine against malaria is thus a majorpriority for global public health. A considerable body of immunologicalresearch over the last twenty years had led to the identification bothof candidate vaccine antigens from the parasite and immunologicalmechanisms on the host that are likely to protect against infection anddisease. However, despite this progress there is still no means ofvaccinating against malaria infection which has been shown to beeffective in field trials.

A major problem has been the identification of a means of inducing asufficiently strong immune response in vaccinated individuals to protectagainst infection and disease. So, although many malaria antigens areknown that might be useful in vaccinating against malaria the problemhas been how to deliver such antigens or fragments of them known asepitopes, which are recognized by cells of the immune system, in a waythat induces a sufficiently strong immune response of a particular type.

It has been known for many years that it is possible to protectindividuals by immunizing them with very large doses of irradiatedmalaria sporozoite given by bites from infected mosquitoes. Althoughthis is a wholly impractical method of mass vaccination it has provideda model for analyzing the immune responses that might be mediatingprotective immunity against sporozoite infection (Nardin and Nussenzweig1993).

A considerable amount of research over the last decade or more hasindicated that a major protective immune response against the earlypre-erythrocytic stage of P. falciparum malaria is mediated by Tlymphocytes of the CD8+ ve type (CD8+ T cells). Such cells have beenshown to mediate protection directly in mouse models of malariainfection (Nardin and Nussenzweig 1993). Such T cells have also beenidentified in individuals naturally exposed to malaria and in volunteersimmunized with irradiated sporozoite (Hill et al. 1991; Aidoo et al.1995; Wizel et al. 1995). There is much indirect evidence that such CD8+T cells are protective against malaria infection and disease in humans(Lalvani et al. 1994).

CD8+ T cells may function in more than one way. The best known functionis the killing or lysis of target cells bearing peptide antigen in thecontext of an MHC class I molecule. Hence these cells are often termedcytotoxic T lymphocytes (CTL). However, another function, perhaps ofgreater protective relevance in malaria infections is the ability ofCD8+ T cells to secrete interferon gamma (IFN-ÿ). Thus assays of lyticactivity and of IFN-ÿ release are both of value in measuring a CD8+ Tcell immune response. In malaria these CD8+ve cells can protect bykilling the parasite at the early intrahepatic stage of malariainfection before any symptoms of disease are produced (Seguin et al.1994).

The agent of fatal human malaria, P. falciparum infects a restrictednumber of host species: humans, chimpanzees and some species of NewWorld monkey. The best non-human model of malaria is the chimpanzeebecause this species is closely related to humans and liver-stageinfection is observed consistently unlike in the monkey hosts (Thomas etal. 1994). Because of the expense and limited availability ofchimpanzees most laboratory studies of malaria are performed in mice,using the rodent malaria species P. berghei or P. yoelii. These lattertwo models are well studied and it has been shown in both that CD8+velymphocytes play a key role in protective immunity against sporozoitechallenge.

Previous studies have assessed a large variety of means of inducing CD8+T cell responses against malaria. Several of these have shown some levelof CD8+ T cell response and partial protection against malaria infectionin the rodent models (e.g. Li et al. 1993; Sedegah et al. 1994; Lanar etal. 1996). However, an effective means of immunizing with subunitvaccines by the induction of sufficiently high levels of CD8+ Tlymphocytes to protect effectively against malaria sporozoite infectionhas not previously been demonstrated.

In recent years improved immune responses generated to potentialvaccines have been sought by varying the vectors used to deliver theantigen. There is evidence that in some instances antibody responses areimproved by using two different vectors administered sequentially asprime and boost. A variety of combinations of prime and boost have beentested in different potential vaccine regimes.

Leong et al. (Vaccines 1995, 327-331) describe immunizing mice firstlyto DNA expressing the influenza haemagglutinin (HA) antigen and thenwith a recombinant fowlpox vector expressing HA. An enhanced antibodyresponse was obtained following boosting.

Richmond et al. (Virology 1997, 230: 265-274) describe attempts to raiseneutralizing antibodies against HIV-1 env using DNA priming andrecombinant vaccinia virus boosting. Only low levels of antibodyresponses were observed with this prime boost regime and the resultswere considered disappointing.

Fuller et al. (Vaccine 1997, 15:924-926 and Immunol Cell Biol 1997,75:389-396) describe an enhancement of antibody responses to DNAimmunization of macaques by using a booster immunization withreplicating recombinant vaccinia viruses. However, this did nottranslate into enhanced protective efficacy as a greater reduction inviral burden and attenuation of CD4 T cell loss was seen in the DNAprimed and boosted animals.

Hodge et al. (Vaccine 1997, 15: 759-768) describe the induction oflymphoproliferative T cell responses in a mouse model for cancer usinghuman carcinoembryonic antigen (CEA) expressed in a recombinant fowl poxvirus (ALVAC). The authors primed an immune response withCEA-recombinant replication competent vaccinia viruses of the Wyeth orWR strain and boosted the response with CEA-recombinant ALVAC. This ledto an increase in T cell proliferation but did not result in enhancedprotective efficacy if compared to three wild type recombinantimmunizations (100% protection), three recombinant ALVAC-CEAimmunizations (70% protection) or WR prime followed by two ALVAC-CEAimmunizations (63% protection).

Thus some studies of heterologous prime-boost combination have foundsome enhancement of antibody and lymphoproliferative responses but nosignificant effect on protective efficacy in an animal model. CD8 Tcells were not measured in these studies. The limited enhancement ofantibody response probably simply reflects the fact that antibodies tothe priming immunogen will often reduce the immunogenicity of a secondimmunization with the same immunogen, while boosting with a differentcarrier will in part overcome this problem. This mechanism would not beexpected to be significantly affected by the order of immunization.

Evidence that a heterologous prime boost immunization regime mightaffect CD8 T cell responses was provided by Li et al. (1993). Theydescribed partial protective efficacy induced in mice against malariasporozoite challenge by administering two live viral vectors, arecombinant replicating influenza virus followed by a recombinantreplicating vaccinia virus encoding a malaria epitope. Reversing theorder of immunization led to loss of all protective efficacy and theauthors suggested that this might be related to infection of liver cellsby vaccinia, resulting in localization of CTLs in the liver to protectagainst the hepatocytic stages of malaria parasites.

Rodrigues et al. (J. Immunol. 1994, 4636-4648) describe immunizing micewith repeated doses of a recombinant influenza virus expressing animmunodominant B cell epitope of the malarial circumsporozoite (CS)protein followed by a recombinant vaccinia virus booster. The use of awild type vaccinia strain and an attenuated but replication-competentvaccinia strain in the booster yielded very similar levels of partialprotection. However the attenuated but replication competent strain wasslightly less immunogenic for priming CD8 T cells than the wild typevaccinia strain.

Murata et al. (Cell. Immunol. 1996, 173: 96-107) reported enhanced CD8 Tcell responses after priming with replicating recombinant influenzaviruses and boosting with a replicating strain of vaccinia virus andsuggested that the partial protection observed in the two earlierstudies was attributable to this enhanced CD8 T cell induction.

Thus these three studies together provide evidence that a boosterimmunization with a replicating recombinant vaccinia virus may enhanceto some degree CD8 T cell induction following priming with a replicatingrecombinant influenza virus. However, there are two limitations to thesefindings in terms of their potential usefulness. Firstly, theimmunogenicity induced was only sufficient to achieve partial protectionagainst malaria and even this was dependent on a highly immunogenicpriming immunization with an unusual replicating recombinant influenzavirus. Secondly, because of the potential and documented side-effects ofusing these replicating viruses as immunogens these recombinant vectorsare not suitable for general human use as vaccines.

Modified vaccinia virus Ankara (MVA) is a strain of vaccinia virus whichdoes not replicate in most cell types, including normal human tissues.MVA was derived by serial passage >500 times in chick embryo fibroblasts(CEF) of material derived from a pox lesion on a horse in Ankara, Turkey(Mayr et al. 1975). It was shown to be replication-impaired yet able toinduce protective immunity against veterinary poxvirus infections (Mayr1976). MVA was used as a human vaccine in the final stages of thesmallpox eradication campaign, being administered by intracutaneous,subcutaneous and intramuscular routes to >120,000 subjects in southernGermany. No significant side effects were recorded, despite thedeliberate targeting of vaccination to high risk groups such as thosewith eczema (Mayr et al. 1978; Stickl et al. 1974; Mahnel et al. 1994;).The safety of MVA reflects the avirulence of the virus in animal models,including irradiated mice and following intracranial administration toneonatal mice. The non-replication of MVA has been correlated with theproduction of proliferative white plaques on chick chorioallantoicmembrane, abortive infection of non-avian cells, and the presence of sixgenomic deletions totaling approximately 30 kb (Meyer et al. 1991). Theavirulence of MVA has been ascribed partially to deletions affectinghost range genes K1L and C7L, although limited viral replication stilloccurs on human TK-143 cells and African Green Monkey CV-1 cells(Altenburger et al. 1989). Restoration of the K1L gene only partiallyrestores MVA host range (Sutter et al. 1994). The host range restrictionappears to occur during viral particle maturation, with only immaturevirions being observed in human HeLa cells on electron microscopy(Sutter et al. 1992). The late block in viral replication does notprevent efficient expression of recombinant genes in MVA. RecombinantMVA expressing influenza nucleoprotein, influenza haemagglutinin, andSIV proteins have proved to be immunogenic and provide varying degreesof protection in animal models, although this has never been ascribed toCD8+ T lymphocytes alone (Sutter et al. 1994, Hirsch et al. 1995; Hirschet al. 1996). Recombinant MVA is considered a promising human vaccinecandidate because of these properties of safety and immunogenicity (Mosset al. 1995). Recombinant MVA containing DNA which codes for foreignantigens is described in U.S. Pat. No. 5,185,146 (Altenburger).

Poxviruses have evolved strategies for evasion of the host immuneresponse that include the production of secreted proteins that functionas soluble receptors for tumor necrosis factor, IL-1ÿ, interferon(IFN)-ÿ/ÿ and IFN-ÿ, which normally have sequence similarity to theextracellular domain of cellular cytokine receptors (Symons et al. 1995;Alcami et al. 1995; Alcami et al. 1992). The most recently describedreceptor of this nature is a chemokine receptor (Graham et al. 1997).These viral receptors generally inhibit or subvert an appropriate hostimmune response, and their presence is associated with increasedpathogenicity. The Il-1ÿ receptor is an exception: its presencediminishes the host febrile response and enhances host survival in theface of infection (Alcami et al. 1996). We have discovered that MVAlacks functional cytokine receptors for interferon ÿ, interferon ÿÿ,Tumor Necrosis Factor and CC chemokines, but it does possess thepotentially beneficial IL-1ÿ receptor. MVA is the only known strain ofvaccinia to possess this cytokine receptor profile, which theoreticallyrenders it safer and more immunogenic than other poxviruses. Anotherreplication-impaired and safe strain of vaccinia known as NYVAC is fullydescribed in Tartaglia et al. (Virology 1992, 188: 217-232).

It has long been recognized that live viruses have some attractivefeatures as recombinant vaccine vectors including a high capacity forforeign antigens and fairly good immunogenicity for cellular immuneresponses (Ellis 1988 new technologies for making vaccines. In:Vaccines. Editors: Plotkin S A and Mortimer E A. W B Saunders,Philadelphia, page 568; Woodrow G C 1977. In: New Generation Vaccines2^(nd) Edition. Editors: Levine M M, Woodrow G C, Kaper J B, Cobon G,page 33). This has led to attempts to attenuate the virulence of suchlive vectors in various ways including reducing their replicationcapacity (Tartaglia J et al. 1992 Virology 188: 217-232). However such areduction in replication reduces the amount of antigen produced by thevirus and thereby would be expected to reduce vaccine immunogenicity.Indeed attenuation of replicating vaccinia strains has previously beenshown to lead to some substantial reductions in antibody responses (LeeM S et al, 1992 J Virology 66: 2617-2630). Similarly the non-replicatingfowlpox vector was found to be less immunogenic for antibody productionand less protective than a replicating wild-type vaccinia strain in arabies study (Taylor J et al. 1991 Vaccine 9: 190-193).

SUMMARY OF THE INVENTION

It has now been discovered that non-replicating and replication-impairedstrains of poxvirus provide vectors which give an extremely goodboosting effect to a primed CTL response. Remarkably, this effect issignificantly stronger than a boosting effect by wild type poxviruses.The effect is observed with malarial and other antigens such as viraland tumor antigens, and is protective as shown in mice and non-humanprimate challenge experiments. Complete rather than partial protectionfrom sporozoite challenge has been observed with the novel immunizationregime.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construct used to express Ty-VLP with the malariaepitope cassette CABDHFE. CTL epitopes are from P. falciparum STARP(sporozoite threonine- and asparagine-rich protein) (st), LSA-1 (liverstage antigen 1) (1s), CSP (circumsporozoite protein) (cp), TRAP(thrombospondin-related adhesive protein) (tr), LSA-3 (liver stageantigen 3) (la) and Exp-1 (exported protein 1) (ex). Helper epitopes arefrom the P. falciparum CS protein, the M. tuberculosis 38 Kd antigen andTetanus Toxoid. NANP is the antibody epitope from CS and AM is theadhesion motif from P. falciparum TRAP (Muller et al 1993). The lengthof the complete string is 229 amino acids.

FIG. 2 shows a schematic outline of the H, M and HM proteins. The barpatterns on the schematic representations of the polyepitope proteinsindicate the origin of the sequences. The positions of individualepitopes and their MHC restrictions are depicted above and below theproteins. Pb is the only epitope derived from the protein of P. berghei.All other epitopes in the M protein originate from proteins of P.falciparum: cs—circumsporozoite protein, st—STARP, Is—LSA-1 and tr—TRAP.BCG—38 kDa protein of M. tuberculosis; TT—tetanus toxin.

FIG. 3 shows malaria CD8 T cell ELISPOT data following differentimmunisation regimes. Results are shown as the number ofpeptide-specific T cells per million splenocytes.

FIGS. 4A-4D show that malaria CD8 T cell ELISPOT (FIGS. 4A and 4C) andCTL levels (FIGS. 4B and 4D) are substantially boosted by a recombinantMVA immunisation following priming with a plasmid DNA encoding the sameantigen. The ELISPOT counts are presented on a logarithmic scale.

FIG. 5 shows the CTL responses induced in BALB/c mice to malaria and HIVepitopes by various immunisation regimes employing plasmid DNA andrecombinant MVA. Levels of specific lysis at various effector to targetratios are shown.

FIG. 6 shows the results of ELISPOT assays performed to measure thelevels of specific CD8+ T cells to the malaria epitope pb9 followingdifferent immunisation regimes. Groups of BALB/c mice (n=3) wereimmunised as indicated (g.g.=gene gun). The time between allimmunisations was 14 days. ELISPOT assays were done two weeks after thelast immunisation.

FIGS. 7A-7E show the CTL responses against influenza NP in differentmouse strains. Mice of different strains were immunised twice two weeksapart with a DNA vaccine V1J-NP encoding for the influenza nucleoprotein(open circles) or primed with the same DNA vaccine and two weeks laterboosted with recombinant MVA expressing influenza virus nucleoprotein(closed circles). The CTL activity was determined in a standard⁵¹Cr-release assay with MHC class I-matched target cells.

FIGS. 8A-8H show CTL responses against different antigens induced indifferent inbred mouse strains. Mice were immunised with two DNA vaccineimmunisations two weeks apart (open circles) or primed with a DNAvaccine and two weeks later boosted with a recombinant MVA expressingthe same antigen (closed circles). The strains and antigens were: FIG.8A, C57BL/6, P. falciparum TRAP; FIG. 8B, DBA/2, E. colib-galactosidase; FIG. 8C, BALB/c, HM epitope string CTL activity againstmalaria peptide (pb9); FIG. 8D, DBA/2, HM epitope string CTL activityagainst pb9; FIG. 8E, BALB/c;,HM epitope string CTL activity against HIVpeptide; FIG. 8F, DBA/2, HM epitope string CTL activity against HIVpeptide; FIG. 8G, BALB/c, tumour epitope string CTL activity againstP1A-derived peptide; and in FIG. 8H, DBA/2, tumour epitope string CTLactivity against P1A-derived peptide. Each curve shows the data for anindividual mouse.

FIGS. 9A-9E show sporozoite-primed CTL responses are substantiallyboosted by MVA. Mice were immunised with: FIG. 9A, two low doses (50+50)of irradiated sporozoites; FIG. 9B, two high doses (300+500) ofsporozoites; FIG. 9D, low-dose sporozoite priming followed by boostingwith MVA.PbCSP; FIG. 9E, high dose sporozoite priming followed byboosting with MVA.PbCSP. CTL responses following immunisation withMVA.PbCSP are shown in FIG. 9C.

FIGS. 10A and 10B show CTL responses primed by plasmid DNA orrecombinant Adenovirus and boosted with MVA. Groups of BALB/c mice (n=3)were primed with plasmid DNA(FIG. 10A) or recombinant Adenovirusexpressing ÿ-galactosidase (FIG. 10B). Plasmid DNA was administeredintramuscularly, MVA intravenously and Adenovirus intradermally.Splenocytes were restimulated with peptide TPHPARIGL [SEQ ID NO: 69] twoweeks after the last immunisation. CTL activity was tested withpeptide-pulsed P815 cells.

FIGS. 11A-11C show CTL responses in BALB/c mice primed with plasmid DNAfollowed by boosting with different recombinant vaccinia viruses.Animals were primed with pTH.PbCSP 50 ÿg/mouse i.m. and two weeks laterboosted with different strains of recombinant vaccina viruses (10⁶ pfuper mouse i.v.) expressing PbCSP. The different recombinant vacciniavirus strains were: FIG. 11A, MVA; FIG. 11B, NYVAC; and WR in Figure C.The frequencies of peptide-specific CD8+ T cells were determined usingthe ELISPOT assay.

FIG. 12 shows frequencies of peptide-specific CD8+ T cells followingdifferent routes of MVA boosting. Results are shown as the number ofspot-forming cells (SFC) per one million splenocytes. Each barrepresents the mean number of SFCs from three mice assayed individually.

FIG. 13 shows the survival rate of the two groups of mice. Sixty daysafter challenge eight out of ten mice were alive in the group immunisedwith the tumour epitopes string.

FIG. 14 shows results of an influenza virus challenge experiment. BALB/cmice were immunised as indicated. GG=gene gun immunisations,im=intramuscular injection, iv=intravenous injection. Survival of theanimals was monitored daily after challenge.

FIGS. 15A-15C show detection of SIV-specific MHC class I-restricted CD8+T cells using tetramers. Each bar represents the percentage of CD8+ Tcells specific for the Mamu-A*01/gag epitope at the indicated timepoint. One percent of CD8 T cells corresponds to about 5000/10⁶peripheral blood lymphocytes.

FIGS. 16A-16C show CTL induction in macaques following DNA/MVAimmunisation. PBMC from three different macaques (CYD, DI and DORIS)were isolated at week 18, 19 and 23 and were restimulated with peptideCTPYDINQM [SEQ ID NO: 54] in vitro. After two restimulations withpeptide CTPYDINQM [SEQ ID NO: 54] the cultures were tested for theirlytic activity on peptide-pulsed autologous target cells.

DETAILED DESCRIPTION OF THE INVENTION

It is an aim of this invention to identify an effective means ofimmunizing against malaria. It is a further aim of this invention toidentify means of immunizing against other diseases in which CD8+ T cellresponses play a protective role. Such diseases include but are notlimited to infection and disease caused by the viruses HIV, herpessimplex, herpes zoster, hepatitis C, hepatitis B, influenza,Epstein-Barr virus, measles, dengue and HTLV-1; by the bacteriaMycobacterium tuberculosis and Listeria sp.; and by the protozoanparasites Toxoplasma and Trypanosoma; and certain forms of cancer e.g.melanoma, cancer of the breast and cancer of the colon.

We describe here a novel method of immunizing that generated very highlevels of CD8+ T cells and was found to be capable of inducingunprecedented complete protection against P. berghei sporozoitechallenge. The same approach was tested in higher primates and found tobe highly immunogenic in this species also, and was found to inducepartial protection against P. falciparum challenge. Induction ofprotective immune responses has also been demonstrated in two additionalmouse models of viral infection and cancer.

We show further than the novel immunization regime that is describedhere is also effective in generating strong CD8+ T cell responsesagainst HIV epitopes. Considerable evidence indicates that thegeneration of such CD8+ T cell responses can be expected to be of valuein prophylactic or therapeutic immunization against this viral infectionand disease (Gallimore et al 1995; Ada 1996). We demonstrate that strongCD8+T cell responses may be generated against epitopes from both HIV andmalaria using an epitope string with sequences from both of thesemicro-organisms. The success in generating enhanced immunogenicityagainst both HIV and malaria epitopes, and also against influenza andtumor epitopes, indicates that this novel immunization regime can beeffective generally against many infectious pathogens and also innon-infectious diseases where the generation of a strong CD8+ T cellresponse may be of value.

A surprising feature of the current invention is the finding of the veryhigh efficacy of non-replicating agents in both priming and particularlyin boosting a CD8+ T cell response. In general the immunogenicity ofCD8+ T cell induction by live replicating viral vectors has previouslybeen found to be higher than for non-replicating agents orreplication-impaired vectors. This is as would be expected from thegreater amount of antigen produced by agents that can replicate in thehost. Here however we find that the greatest immunogenicity andprotective efficacy is surprisingly observed with non-replicatingvectors. The latter have an added advantage for vaccination in that theyare in general safer for use in humans than replicating vectors.

The present invention provides in one aspect a kit for generating aprotective CD8+ T cell immune response against at least one targetantigen, which kit comprises:

-   -   (i) a priming composition comprising a source of one or more        CD8+ T cell epitopes of the target antigen, together with a        pharmaceutically acceptable carrier; and    -   (ii) a boosting composition comprising a source of one or more        CD8+ T cell epitopes of the target antigen, including at least        one CD8+ T cell epitope which is the same as a CD8+ T cell        epitope of the priming composition, wherein the source of CD8+ T        cell epitopes is a non-replicating or replication-impaired        recombinant poxvirus vector, together with a pharmaceutically        acceptable carrier; with the proviso that if the source of        epitopes in (i) is a viral vector, the viral vector in (ii) is        derived from a different virus.

In another aspect the invention provides a method for generating aprotective CD8+ T cell immune response against at least one targetantigen, which method comprises administering at least one dose ofcomponent (i), followed by at least one dose of component (ii) of thekit according to the invention.

Preferably, the source of CD8+ T cell epitopes in (i) in the methodaccording to the invention is a non-viral vector or a non-replicating orreplication-impaired viral vector, although replicating viral vectorsmay be used.

Preferably, the source of CD8+ T cell epitopes in (i) is not a poxvirusvector, so that there is minimal cross-reactivity between the primer andthe booster.

In one preferred embodiment of the invention, the source of CD8+ T cellepitopes in the priming composition is a nucleic acid, which may be DNAor RNA, in particular a recombinant DNA plasmid. The DNA or RNA may bepackaged, for example in a lysosome, or it may be in free form.

In another preferred embodiment of the invention, the source of CD8+ Tcell epitopes in the priming composition is a peptide, polypeptide,protein, polyprotein or particle comprising two or more CD8+ T cellepitopes, present in a recombinant string of CD8+ T cell epitopes or ina target antigen. Polyproteins include two or more proteins which may bethe same, or preferably different, linked together. Particularlypreferred in this embodiment is a recombinant proteinaceous particlesuch as a Ty virus-like particle (VLP) (Burns et al. Molec. Biotechnol.1994, 1: 137-145).

Preferably, the source of CD8+ T cell epitopes in the boostingcomposition is a vaccinia virus vector such as MVA or NYVAC. Mostpreferred is the vaccinia strain modified virus ankara (MVA) or a strainderived therefrom. Alternatives to vaccinia vectors include avipoxvectors such as fowlpox or canarypox vectors. Particularly suitable asan avipox vector is a strain of canarypox known as ALVAC (commerciallyavailable as Kanapox), and strains derived therefrom.

Poxvirus genomes can carry a large amount of heterologous geneticinformation. Other requirements for viral vectors for use in vaccinesinclude good immunogenicity and safety. MVA is a replication-impairedvaccinia strain with a good safety record. In most cell types and normalhuman tissues, MVA does not replicate; limited replication of MVA isobserved in a few transformed cell types such as BHK21 cells. It has nowbeen shown, by the results described herein, that recombinant MVA andother non-replicating or replication-impaired strains are surprisinglyand significantly better than conventional recombinant vaccinia vectorsat generating a protective CD8+ T cell response, when administered in aboosting composition following priming with a DNA plasmid, a recombinantTy-VLP or a recombinant adenovirus.

It will be evident that vaccinia virus strains derived from MVA, orindependently developed strains having the features of MVA which makeMVA particularly suitable for use in a vaccine, will also be suitablefor use in the invention.

MVA containing an inserted string of epitopes (MVA-HM, which isdescribed in the Examples) has been deposited at the European Collectionof Animal Cell Cultures, CAMR, Salisbury, Wiltshire SP4 0JG, UK underaccession no. V97060511 on 5 Jun. 1997.

The term “non-replicating” or “replication-impaired” as used hereinmeans not capable of replication to any significant extent in themajority of normal mammalian cells or normal human cells. Viruses whichare non-replicating or replication-impaired may have become so naturally(i.e. they may be isolated as such from nature) or artificially e.g. bybreeding in vitro or by genetic manipulation, for example deletion of agene which is critical for replication. There will generally be one or afew cell types in which the viruses can be grown, such as CEF cells forMVA.

Replication of a virus is generally measured in two ways: 1) DNAsynthesis and 2) viral titre. More precisely, the term “non-replicatingor replication-impaired” as used herein and as it applies to poxvirusesmeans viruses which satisfy either or both of the following criteria:

-   -   1) exhibit a 1 log (10 fold) reduction in DNA synthesis compared        to the Copenhagen strain of vaccinia virus in MRC-5 cells (a        human cell line);    -   2) exhibit a 2 log reduction in viral titre in HELA cells (a        human cell line) compared to the Copenhagen strain of vaccinia        virus.

Examples of poxviruses which fall within this definition are MVA, NYVACand avipox viruses, while a virus which falls outside the definition isthe attenuated vaccinia strain M7.

Alternative preferred viral vectors for use in the priming compositionaccording to the invention include a variety of different viruses,genetically disabled so as to be non-replicating orreplication-impaired. Such viruses include for example non-replicatingadenoviruses such as E1 deletion mutants. Genetic disabling of virusesto produce non-replicating or replication-impaired vectors has beenwidely described in the literature (e.g. McLean et al. 1994).

Other suitable viral vectors for use in the priming composition arevectors based on herpes virus and Venezuelan equine encephalitis virus(VEE) (Davies et al. 1996). Suitable bacterial vectors for priminginclude recombinant BCG and recombinant Salmonella and Salmonellatransformed with plasmid DNA (Darji A et al. 1997 Cell 91: 765-775).

Alternative suitable non-viral vectors for use in the primingcomposition include lipid-tailed peptides known as lipopeptides,peptides fused to carrier proteins such as KLH either as fusion proteinsor by chemical linkage, whole antigens with adjuvant, and other similarsystems. Adjuvants such as QS21 or SBAS2 (Stoute J A et al. 1997 N EnglJ Medicine 226: 86-91) may be used with proteins, peptides or nucleicacids to enhance the induction of T cell responses. These systems aresometimes referred to as “immunogens” rather than “vectors”, but theyare vectors herein in the sense that they carry the relevant CD8+ T cellepitopes.

There is no reason why the priming and boosting compositions should notbe identical in that they may both contain the priming source of CD8+ Tcell epitopes as defined in (i) above and the boosting source of CD8+ Tcell epitopes as defined in (ii) above. A single formulation which canbe used as a primer and as a booster will simplify administration. Theimportant thing is that the primer contains at least the priming sourceof epitopes as defined in (i) above and the booster contains at leastthe boosting source of epitopes as defined in (ii) above.

The CD8+ T cell epitopes either present in, or encoded by the primingand boosting compositions, may be provided in a variety of differentforms, such as a recombinant string of one or two or more epitopes, orin the context of the native target antigen, or a combination of both ofthese. CD8+ T cell epitopes have been identified and can be found in theliterature, for many different diseases. It is possible to designepitope strings to generate a CD8+ T cell response against any chosenantigen that contains such epitopes. Advantageously, the epitopes in astring of multiple epitopes are linked together without interveningsequences so that unnecessary nucleic acid and/or amino acid material isavoided. In addition to the CD8+ T cell epitopes, it may be preferableto include one or more epitopes recognized by T helper cells, to augmentthe immune response generated by the epitope string. Particularlysuitable T helper cell epitopes are ones which are active in individualsof different HLA types, for example T helper epitopes from tetanus(against which most individuals will already be primed). A usefulcombination of three T helper epitopes is employed in the examplesdescribed herein. It may also be useful to include B cell epitopes forstimulating B cell responses and antibody production.

The priming and boosting compositions described may advantageouslycomprise an adjuvant. In particular, a priming composition comprising aDNA plasmid vector may also comprise granulocyte macrophage-colonystimulating factor (GM-CSF), or a plasmid encoding it, to act as anadjuvant; beneficial effects are seen using GM-CSF in polypeptide form.

The compositions described herein may be employed as therapeutic orprophylactic vaccines. Whether prophylactic or therapeutic immunizationis the more appropriate will usually depend upon the nature of thedisease. For example, it is anticipated that cancer will be immunizedagainst therapeutically rather than before it has been diagnosed, whileanti-malaria vaccines will preferably, though not necessarily be used asa prophylactic.

The compositions according to the invention may be administered via avariety of different routes. Certain routes may be favoured for certaincompositions, as resulting in the generation of a more effectiveresponse, or as being less likely to induce side effects, or as beingeasier for administration. The present invention has been shown to beeffective with gene gun delivery, either on gold beads or as a powder.

In further aspects, the invention provides:

-   -   a method for generating a protective CD8+ T cell immune response        against a pathogen or tumor, which method comprises        administering at least one dose of a recombinant DNA plasmid        encoding at least one CD8+ T cell epitope or antigen of the        pathogen or cancer, followed by at least one dose of a        non-replicating or replication-impaired recombinant pox virus        encoding the same epitope or antigen;    -   a method for generating a protective CD8+ T cell immune response        against a pathogen or tumor, which method comprises        administering at least one dose of a recombinant protein or        particle comprising at least one epitope or antigen of the        pathogen or cancer, followed by at least one dose of a        recombinant MVA vector encoding the same epitope or antigen;    -   the use of a recombinant non-replicating or replication-impaired        pox virus vector in the manufacture of a medicament for boosting        a CD8+ T cell immune response;    -   the use of an MVA vector in the manufacture of a medicament for        boosting a CD8+ T cell immune response;    -   a medicament for boosting a primed CD8+ T cell response against        at least one target antigen or epitope, comprising a source of        one or more CD8+ T cell epitopes of the target antigen, wherein        the source of CD8+ T cell epitopes is a non-replicating or a        replication-impaired recombinant poxvirus vector, together with        a pharmaceutically acceptable carrier; and    -   the priming and/or boosting compositions described herein, in        particulate form suitable for delivery by a gene gun; and        methods of immunization comprising delivering the compositions        by means of a gene gun.

Also provided by the invention are: the epitope strings describedherein, including epitope strings comprising the amino acid sequenceslisted in table 1 and table 2; recombinant DNA plasmids encoding theepitope strings; recombinant Ty-VLPs comprising the epitope strings; arecombinant DNA plasmid or non-replicating or replication impairedrecombinant pox virus encoding the P. falciparum antigen TRAP; and arecombinant polypeptide comprising a whole or substantially wholeprotein antigen such as TRAP and a string of two or more epitopes insequence such as CTL epitopes from malaria.

Example Formulations and Immunization Protocols Formulation 1

-   Priming Composition: DNA plasmid 1 mg/ml in PBS-   Boosting Composition: Recombinant MVA, 10⁸ ffu in PBS-   Protocol: Administer two doses of 1 mg of priming composition, i.m.,    at 0 and 3 weeks followed by two doses of booster intradermally at 6    and 9 weeks.

Formulation 2

-   Priming Composition: Ty-VLP 500ÿg in PBS-   Boosting Composition: MVA, 10⁸ ffu in PBS-   Protocol: Administer two doses of priming composition, i.m., at 0    and 3 weeks, then 2 doses of booster at 6 and 9 weeks. For tumor    treatment, MVA is given i.v. as one of most effective routes.

Formulation 3

-   Priming Composition: Protein 500ÿg+adjuvant (QS-21)-   Boosting Composition: Recombinant MVA, 10⁸ ffu in PBS-   Protocol: Administer two doses of priming composition at 0 and 3    weeks and 2 doses of booster i.d. at 6 and 9 weeks.

Formulation 4

-   Priming Composition: Adenovirus vector, 10⁹ pfu in PBS-   Boosting Composition: Recombinant MVA, 10⁸ ffu in PBS-   Protocol: Administer one or two doses of priming composition    intradermally at 0 and 3 weeks and two doses of booster i.d. at 6    and 9 weeks.    The above doses and protocols may be varied to optimise protection.    Doses may be given between for example, 1 to 8 weeks apart rather    than 2 weeks apart.

The invention will now be further described in the examples whichfollow.

Examples Example 1 Materials and Methods Generation of the EpitopeStrings

The malaria epitope string was made up of a series of cassettes eachencoding three epitopes as shown in Table 1, with restriction enzymesites at each end of the cassette. Each cassette was constructed fromfour synthetic oligonucleotides which were annealed together, ligatedinto a cloning vector and then sequenced to check that no errors hadbeen introduced. Individual cassettes were then joined together asrequired. The BamHI site at the 3′ end of cassette C was fused to theBglII site at the 5′ end of cassette A, destroying both restrictionenzyme sites and encoding a two amino acid spacer (GS) between the twocassettes. Cassettes B, D and H were then joined to the string in thesame manner. A longer string containing CABDHFE was also constructed inthe same way.

TABLE 1 CTL Epitopes of the Malaria (M) String Amino acid HLA CassetteEpitope Sequence DNA sequence Type restriction A Ls8 KPNDKSLYAAGCCGAACGACAAGTCCTTGTAT CTL B35 SEQ ID NO.: 2 SEQ ID NO.: 1 Cp26KPKDELDY AAACCTAAGGACGAATTGGACTAC CTL B35 SEQ ID NO.: 4 SEQ ID NO.: 3Ls6 KPIVQYDNF AAGCCAATCGTTCAATACGACAACTTC CTL B53 SEQ ID NO.: 6 SEQ IDNO.: 5 B Tr42/43 ASKNKEKALII GCCTCCAAGAACAAGGAAAAGGCTTTGATCATC CTL B8SEQ ID NO.: 8 SEQ ID NO.: 7 Tr39 GIAGGLALL GGTATCGCTGGTGGTTTGGCCTTGTTGCTL A2.1 SEQ ID NO.: 10 SEQ ID NO.: 9 Cp6 MNPNDPNRNVATGAACCCTAATGACCCAAACAGAAACGTC CTL B7 SEQ ID NO.: 12 SEQ ID NO.: 11 CSt8 MINAYLDKL ATGATCAACGCCTACTTGGACAAGTTG CTL A2.2 SEQ ID NO.: 14 SEQ IDNO.: 13 Ls50 ISKYEDEI ATCTCCAAGTACGAAGACGAAATC CTL B17 SEQ ID NO.: 16SEQ ID NO.: 15 Pb9 SYIPSAEKI TCCTACATCCCATCTGCCGAAAAGATC CTL mouse SEQID NO.: 18 SEQ ID NO.: 17 H2-K^(d) D Tr26 HLGNVKYLVCACTTGGGTAACGTTAAGTACTTGGTT CTL A2.1 SEQ ID NO.: 20 SEQ ID NO.: 19 Ls53KSLYDEHI AAGTCTTTGTACGATGAACACATC CTL B58 SEQ ID NO.: 22 SEQ ID NO.: 21Tr29 LLMDCSGSI TTATTGATGGACTGTTCTGGTTCTATT CTL A2.2 SEQ ID NO.: 24 SEQID NO.: 23 E NANP NANPNANPNANPN AACGCTAATCCAAACGCAAATCCGAACGC B cell ANPCAATCCTAACGCGAATCCC SEQ ID NO.: 26 SEQ ID NO.: 25 TRAP AM DEWSPCSVTCGKGACGAATGGTCTCCATGTTCTGTCACTTGT Heparin GTRSRKREGGTAAGGGTACTCGCTCTAGAAAGAGAGAA binding SEQ ID NO.: 28 SEQ ID NO.: 27motif F Cp39 YLNKIQNSL TACTTGAACAAAATTCAAAACTCTTTG CTL A2.1 SEQ ID NO.:30 SEQ ID NO.: 29 La72 MEKLKELEK ATGGAAAAGTTGAAAGAATTGGAAAAG CTL B8 SEQID NO.: 32 SEQ ID NO.: 31 ex23 ATSVLAGL GCTACTTCTGTCTTGGCTGGTTTG CTL B58SEQ ID NO.: 34 SEQ ID NO.: 33 H CSP DPNANPNVDPNANPGACCCAAACGCTAACCCAAACGTTGACCC T Universal NV AAACGCCAACCCAAACGTC helperepitopes SEQ ID NO.: 36 SEQ ID NO.: 35 BCG QVHFQPLPPAVVCAAGTTCACTTCCAACCATTGCCTCCGGC T KL CGTTGTCAAGTTG helper SEQ ID NO.: 38SEQ ID NO.: 37 TT QFIKANSKFIGITE CAATTCATCAAGGCCAACTCTAAGTTCAT T SEQ IDNO.: 40 CGGTATCACCGAA helper SEQ ID NO.: 39

Table 1

Sequences included in the malaria epitope string. Each cassette consistsof the epitopes shown above, in the order shown, with no additionalsequence between epitopes within a cassette. A BglII site was added atthe 5′ end and a BamHI site at the 3′ end, such that between cassettesin an epitope string the BamHI/BglII junction encodes GS. All epitopesare from P. falciparum antigens except for pb9 (P. berghei), BCG (M.tuberculosis) and TT (Tetanus). The amino acid and DNA sequences shownin the table have SEQ ID NOS. 1 to 40 in the order in which they appear.

FIG. 1 shows the construct used to express Ty-VLP with the malariaepitope cassette CABDHFE. CTL epitopes are from P. falciparum STARP(sporozoite threonine- and asparagine-rich protein) (st), LSA-1 (liverstage antigen 1) (1s), CSP (circumsporozoite protein) (cp), TRAP(thrombospondin-related adhesive protein) (tr), LSA-3 (liver stageantigen 3) (la) and Exp-1 (exported protein 1) (ex). Helper epitopes arefrom the P. falciparum CS protein, the M. tuberculosis 38 Kd antigen andTetanus Toxoid. NANP is the antibody epitope from CS and AM is theadhesion motif from P. falciparum TRAP (Muller et al 1993). The lengthof the complete string is 229 amino acids as shown in the table 1legend, with the amino acid sequence:

[SEQ ID NO: 41] MINAYLDKLISKYEDEISYIPSAEKIGSKPNDKSLYKPKDELDYKPIVQYDNFGSASKNKEKALIIGIAGGLALLMNPNDPNRNVGSHLGNVKYLVKSLYDEHILLMDCSGSIGSDPNANPNVDPNANPNVQVHFQPLPPAVVKLQFIKANSKFIGITEGSYLNKIQNSLMEKLKELEKATSVLAGLGSNANPNANPNANPNANPDEWSPCSVTCGKGTRSRKREGSGK.

The HIV epitope string was also synthesised by annealingoligonucleotides. Finally the HIV and malaria epitope strings were fusedby joining the BamHI site at the 3′ end of the HIV epitopes to the BglIIsite at the 5′ end of cassettes CAB to form the HM string (Table 2).

TABLE 2 CTL Epitopes of the HIV/SIV Epitope String Epitope RestrictionOrigin YLKDQQLL (SEQ ID NO.: 42) A24, B8 HIV-1 gp41 ERYLKDQQL (SEQ IDNO.: 43) B14 HIV-1 gp41 EITPIGLAP (SEQ ID NO.: 44) Mamu-B*01 SIV envPPIPVGEIY (SEQ ID NO.: 45) B35 HIV-1 p24 GEIYKRWII (SEQ ID NO.: 46) B8HIV-1 p24 KRWIILGLNK (SEQ ID NO.: 47) B*2705 HIV-1 p24 IILGLNKIVR (SEQID NO.: 48) A33 HIV-1 p24 LGLNKIVRMY (SEQ ID NO.: 49) Bw62 HIV-1 p24YNLTMKCR (SEQ ID NO.: 50) Mamu-A*02 SIV env RGPGRAFVTI (SEQ ID NO.: 51)A2, H-2Dd HIV-1 gp120 GRAFVTIGK (SEQ ID NO.: 52) B*2705 HIV-1 gp120TPYDINQML (SEQ ID NO.: 53) B53 HIV-2 gag CTPYDINQM (SEQ ID NO.: 54)Mamu-A*01 SIV gag RPQVPLRPMTY (SEQ ID NO.: 55) B51 HIV-1 nef QVPLRPMTYK(SEQ ID NO.: 56) A*0301, A11 HIV-1 nef VPLRPMTY (SEQ ID NO.: 57) B35HIV-1 nef AVDLSHFLK (SEQ ID NO.: 58) A11 HIV-1 nef DLSHFLKEK (SEQ IDNO.: 59) A*0301 HIV-1 nef FLKEKGGL (SEQ ID NO.: 60) B8 HIV-1 nefILKEPVHGV (SEQ ID NO.: 61) A*0201 HIV-1 po1 ILKEPVHGVY (SEQ ID NO.: 62)Bw62 HIV-1 pol HPDIVIYQY (SEQ ID NO.: 63) B35 HIV-1 pol VIYQYMDDL (SEQID NO.: 64) A*0201 HIV-1 p01

Table 2

Sequences of epitopes from HIV or SIV contained in the H epitope stringand assembled as shown in FIG. 2. The amino acids in the table have SEQID NOS: 42 to 64 in the order in which they appear.

FIG. 2 shows a schematic outline of the H, M and HM proteins. The barpatterns on the schematic representations of the polyepitope proteinsindicate the origin of the sequences (see tables 1 and 2). The positionsof individual epitopes and their MHC restrictions are depicted above andbelow the proteins. Pb is the only epitope derived from the protein ofP. berghei. All other epitopes in the M protein originate from proteinsof P. falciparum: cs—circumsporozoite protein, st—STARP, Is—LSA-1 andtr—TRAP. BCG—38 kDa protein of M. tuberculosis; TT—tetanus toxin.

For the anti-tumour vaccine an epitope string containing CTL epitopeswas generated, similar to the malaria and HIV epitope string. In thistumour epitope string published murine CTL epitopes were fused togetherto create the tumour epitope string with the amino acid sequence:MLPYLGWLVF-AQHPNAELL-KHYLFRNL-SPSYVYHQF-IPNPLLGLD [SEQ ID NO: 65]. CTLepitopes shown here were fused together. The first amino acid methioninewas introduced to initiate translation.

Ty Virus-Like Particles (Vlps)

The epitope string containing cassette CABDH was introduced into a yeastexpression vector to make a C-terminal in-frame fusion to the TyAprotein. When TyA or TyA fusion proteins are expressed in yeast fromthis vector, the protein spontaneously forms virus like particles whichcan be purified from the cytoplasm of the yeast by sucrose gradientcentrifugation. Recombinant Ty-VLPs were prepared in this manner anddialysed against PBS to remove the sucrose before injection (c.f. Laytonet al. 1996).

Replication-defective recombinant Adenovirus with a deletion of the E1genes was used in this study (McGrory et al, 1988). The Adenovirusexpressed E. coli ÿ-galactosidase under the control of a CMV IEpromoter. For immunisations, 10⁷ pfu of virus were administeredintradermally into the ear lobe.

Peptides were purchased from Research Genetics (USA), dissolved at 10mg/ml in DMSO (Sigma) and further diluted in PBS to 1 mg/ml. Peptidescomprising CTL epitopes that were used in the experiments describedherein are listed in table 3.

TABLE 3 Sequence of CTL Peptide Epitopes MHC restric- sequence Antigention LPYLGWLVF (SEQ ID NO.: 66) P1A tumour L^(d) antigen SYIPSAEKI (SEQID NO.: 67) P. berghei CSP K^(d) RGPGRAFVTI (SEQ ID NO.: 68) HIV gagD^(d) TPHPARIGL (SEQ ID NO.: 69) E. coli b- L^(d) galactosidaseTYQRTRALV (SEQ ID NO.: 70) Influenza A K^(d) virus NP SDYEGRLI (SEQ IDNO.: 71) Influenza A K^(k) virus NP ASNENMETM (SEQ ID NO.: 72) InfluenzaA D^(b) virus NP INVAFNRFL (SEQ ID NO.: 73) P. falciparum K^(b) TRAP

The amino acid sequences in Table 3 have SEQ ID NOS: 66 to 73, in theorder in which they appear in the Table.

Plasmid DNA Constructs

A number of different vectors were used for constructing DNA vaccines.Plasmid pTH contains the CMV IE promoter with intron A, followed by apolylinker to allow the introduction of antigen coding sequences and thebovine growth hormone transcription termination sequence. The plasmidcarries the ampicillin resistance gene and is capable of replication inE. coli but not mammalian cells. This was used to make DNA vaccinesexpressing each of the following antigens: P. berghei TRAP, P. bergheiCS, P. falciparum TRAP, P. falciparum LSA-1 (278 amino acids of the Cterminus only), the epitope string containing cassettes CABDH and the HMepitope string (HIV epitopes followed by cassettes CAB). Plasmid pSG2 issimilar to pTH except for the antibiotic resistance gene. In pSG2 theampicillin resistance gene of pTH has been replaced by a kanamycinresistance gene. pSG2 was used to to make DNA vaccines expressing thefollowing antigens: P. berghei PbCSP, a mouse tumour epitope string, theepitope string containing cassettes CABDH and the HM epitope string.Plasmid V1J-NP expresses influenza nucleoprotein under the control of aCMV IE promoter. Plasmids CMV-TRAP and CMV-LSA-1 are similar to pTH.TRAPand pTH. LSA-1 but do not contain intron A of the CMV promoter. PlasmidsRSV.TRAP and RSV.LSA-1 contain the RSV promoter, SV40 transcriptiontermination sequence and are tetracycline resistant. For induction ofÿ-galactosidase-specific CTL plasmid pcDNA3/His/LacZ (Invitrogen) wasused. All DNA vaccines were prepared from E. coli strain DH5ÿ usingQiagen plasmid purification columns.

Generation of Recombinant Vaccinia Viruses

Recombinant MVAs were made by first cloning the antigen sequence into ashuttle vector with a viral promoter such as the plasmid pSC11(Chakrabarti et al. 1985; Morrison et al. 1989). P. berghei CS and P.falciparum TRAP, influenza nucleoprotein and the HM and mouse tumourepitope polyepitope string were each expressed using the P7.5 promoter(Mackett et al. 1984), and P. berghei TRAP was expressed using thestrong synthetic promoter (SSP; Carroll et al. 1995). The shuttlevectors, pSC11 or pMCO3 were then used to transform cells infected withwild-type MVA so that viral sequences flanking the promoter, antigencoding sequence and marker gene could recombine with the MVA and producerecombinants. Recombinant viruses express the marker gene (ÿglucuronidase or ÿ galactosidase) allowing identification of plaquescontaining recombinant virus. Recombinants were repeatedly plaquepurified before use in immunisations. The recombinant NYVAC-PbCSPvaccinia was previously described (Lanar et al. 1996). The wild type orWestern Reserve (WR) strain of recombinant vaccinia encoding PbCSP wasdescribed previously (Satchidanandam et al. 1991).

Cells and Culture Medium

Murine cells and Epstein-Barr virus transformed chimpanzee and macaque Bcells (BCL) were cultured in RPMI supplemented with 10% heat inactivatedfetal calf serum (FCS). Splenocytes were restimulated with the peptidesindicated (final concentration 1 ÿg/ml) in MEM medium with 10% FCS, 2 mMglutamine, 50 U/ml penicillin, 50 ÿM 2-mercaptoethanol and 10 mM HepespH7.2 (Gibco, UK).

Animals

Mice of the strains indicated, 6-8 weeks old were purchased from HarlanOlac (Shaws Farm, Blackthorn, UK). Chimpanzees H1 and H2 were studied atthe Biomedical Primate Research Centre at Rijswick, The Netherlands.Macaques were studied at the University of Oxford.

Immunisations

Plasmid DNA immunisations of mice were performed by intramuscularimmunisation of the DNA into the musculus tibialis under anaesthesia.Mouse muscle was sometimes pre-treated with 50 ÿl of 1 mM cardiotoxin(Latoxan, France) 5-9 days prior to immunisation as described by Daviset al (1993), but the presence or absence of such pre-treatment was notfound to have any significant effect on immunogenicity or protectiveefficacy. MVA immunisation of mice was performed by either intramuscular(i.m.), intravenous (into the lateral tail vein) (i.v.), intradermal(i.d.), intraperitoneal (i.p.) or subcutaneous (s.c.) immunisation.Plasmid DNA and MVA immunisation of the chimpanzees H1 and H2 wasperformed under anaesthesia by intramuscular immunisation of legmuscles. For these chimpanzee immunisations the plasmid DNA wasco-administered with 15 micrograms of human GM-CSF as an adjuvant.Recombinant MVA administration to the chimpanzees was by intramuscularimmunisation under veterinary supervision. Recombinant human GM-CSF waspurchased from Sandoz (Camberley, UK). For plasmid DNA immunisationsusing a gene gun, DNA was precipitated onto gold particles. Forintradermal delivery, two different types of gene guns were used, theAcell and the Oxford Bioscience device (PowderJect Pharmaceuticals,Oxford, UK).

Elispot Assays

CD8+ T cells were quantified in the spleens of immunised mice without invitro restimulation using the peptide epitopes indicated and the ELISPOTassay as described by Miyahara et al (1993). Briefly, 96-wellnitrocellulose plates (Miliscreen MAHA, Millipore, Bedford UK) werecoated with 15 ÿg/ml of the anti-mouse interferon-ÿ monoclonal antibodyR4 (EACC) in 50 ÿl of phosphate-buffered saline (PBS). After overnightincubation at 4° C. the wells were washed once with PBS and blocked for1 hour at room temperature with 100 ÿl RPMI with 10% FCS. Splenocytesfrom immunised mice were resuspended to 1×10⁷ cells/ml and placed induplicate in the antibody coated wells and serially diluted. Peptide wasadded to each well to a final concentration of 1 ÿg/ml. Additional wellswithout peptide were used as a control for peptide-dependence ofinterferon-ÿ secretion. After incubation at 37° C. in 5% CO₂ for 12-18hours the plates were washed 6 times with PBS and water. The wells werethen incubated for 3 hours at room temperature with a solution of 1ÿg/ml of biotinylated anti-mouse interferon-ÿ monoclonal antibody XMG1.2(Pharmingen, CA, USA) in PBS. After further washes with PBS, 50 ÿl of a1 ÿg/ml solution of streptavidin-alkaline-phosphatase polymer (Sigma)was added for 2 hours at room temperature. The spots were developed byadding 50 ÿl of an alkaline phosphatase conjugate substrate solution(Biorad, Hercules, Calif., USA). After the appearance of spots thereaction was stopped by washing with water. The number of spots wasdetermined with the aid of a stereomicroscope.

ELISPOT assays on the chimpanzee peripheral blood lymphocytes wereperformed using a very similar method employing the assay and reagentsdeveloped to detect human CD8 T cells (Mabtech, Stockholm).

CTL Assays

CTL assays were performed using chromium labelled target cells asindicated and cultured mouse spleen cells as effector cells as describedby Allsopp et al. (1996). CTL assays using chimpanzee or macaque cellswere performed as described for the detection of human CTL by Hill etal. (1992) using EBV-transformed autologous chimpanzee chimpanzee ormacaque B cell lines as target cells.

P. Berghei Challenge

Mice were challenged with 2000 (BALB/c) or 200 (C57BL/6) sporozoites ofthe P. berghei ANKA strain in 200 ml RPMI by intravenous inoculation asdescribed (Lanar et al. 1996). These sporozoites were dissected from thesalivary glands of Anopheles stephensi mosquitoes maintained at 18° C.for 20-25 days after feeding on infected mice. Blood-stage malariainfection, indicating a failure of the immunisation, was detected byobserving the appearance of ring forms of P. berghei in Giemsa-stainedblood smears taken at 5-12 days post-challenge.

P. Falciparum Challenge

The chimpanzees were challenged with 20,000 P. falciparum sporozoites ofthe NF54 strain dissected from the salivary glands of Anopheles gambiaemosquitoes, by intravenous inoculation under anaesthesia. Blood samplesfrom these chimpanzees were examined daily from day 5 after challenge bymicroscopy and parasite culture, in order to detect the appearance oflow levels of P. falciparum parasites in the peripheral blood.

Mice were challenged with 1×10⁵ P815 cells in 200 ÿl of PBS byintravenous inoculation. Animals were monitored for survival.

Influenza Virus Challenges

Mice were challenged with 100 haemagglutinating units (HA) of influenzavirus A/PR/8/34 by intranasal inoculation. Following challenge theanimals were weighed daily and monitored for survival.

Determining Peptide Specific CTL Using Tetramers

Tetrameric complexes consisting of Mamu-A*01-heavy chain and ÿ_(2.)microglobulin were made as described by Ogg et al (1998). DNA coding forthe leaderless extracellular portion of the Mamu-A*01 MHC class I heavychain was PCR-amplified from cDNA using 5′primer MamuNdeI: 5′-CCT GACTCA GAC CAT ATG GGC TCT CAC TCC ATG [SEQ ID NO: 74] and 3′ primer:5′-GTG ATA AGC TTA ACG ATG ATT CCA CAC CAT TTT CTG TGC ATC CAG AAT ATGATG CAG GGA TCC CTC CCA TCT CAG GGT GAG GGG C [SEQ ID NO: 75]. Theformer primer contained a NdeI restriction site, the latter included aHindIII site and encoded for the bioinylation enzyme BirA substratepeptide. PCR products were digested with NdeI and HindIII and ligatedinto the same sites of the polylinker of bacterial expression vectorpGMT7. The rhesus monkey gene encoding a leaderless ÿ_(2.)microglobulinwas PCR amplifed from a cDNA clone using primers B2MBACK: 5′-TCA GAC CATATG TCT CGC TCC GTG GCC [SEQ ID NO: 76] and B2MFOR: 5═-TCA GAC AAG CTTTTA CAT GTC TCG ATC CCA C [SEQ ID NO: 77] and likewise cloned into theNdeI and HindIII sites of pGMT7. Both chains were expressed in E. colistrain BL-21, purified from inclusion bodies, refolded in the presenceof peptide CTPYDINQM [SEQ ID NO: 54], biotinylated using the BirA enzyme(Avidity) and purified with FPLC and monoQ ion exchange columns. Theamount of biotinylated refolded MHC-peptide complexes was estimated inan ELISA assay, whereby monomeric complexes were first captured byconformation sensitive monoclonal antibody W6/32 and detected byalkaline phosphatase (AP)-conjugated streptavidin (Sigma) followed bycolorimetric substrate for AP. The formation of tetrameric complexes wasinduced by addition of phycoerythrin (PE)-conjugated streptavidin(ExtrAvidin; Sigma) to the refolded biotinylated monomers at a molarratio of MHC-peptide: PE-streptavidin of 4:1. The complexes were storedin the dark at 4° C. These tetramers were used to analyse the frequencyof Mamu-A*01/gag-specific CD8+ T cells in peripherial blood lymphocytes(PBL) of immunised macaques.

Example 2

Previous studies of the induction of CTL against epitopes in thecircumsporozoite (CS) protein of Plasmodium berghei and Plasmodiumyoelii have shown variable levels of CTL induction with differentdelivery systems. Partial protection has been reported with plasmid DNA(Sedegah et al. 1994), influenza virus boosted by replicating vacciniavirus (Li et al. 1991), adenovirus (Rodrigues et al 1997) and particledelivery systems (Schodel et al. 1994). Immunisation of miceintramuscularly with 50 micrograms of a plasmid encoding the CS proteinproduced moderate levels of CD8+ cells and CTL activity in the spleensof these mice after a single injection (FIGS. 3, 4A-4D).

For comparison groups of BALB/c mice (n=5) were injected intravenouslywith 10⁶ ffu/pfu of recombinant vaccinia viruses of different strains(WR, NYVAC and MVA) all expressing P. berghei CSP. The frequencies ofpeptide-specific CD8+ T cells were measured 10 days later in an ELISPOTassay. MVA.PbCSP induced 181±48, NYVAC 221±27 and WR 94±19(mean±standard deviation) peptide-specific CD8+ T cells per millionsplenocytes. These results show that surprisingly replication-impairedvaccinia viruses are superior to replicating strains in priming a CD8+ Tcell response. We then attempted to boost these moderate CD8+ T cellresponses induced by priming with either plasmid DNA or MVA usinghomologous or heterologous vectors. A low level of CD8+ T cells wasobserved after two immunisations with CS recombinant DNA vaccine alone,the recombinant MVA vaccine alone or the recombinant MVA followed byrecombinant DNA (FIG. 3). A very much higher level of CD8+ T cells wasobserved by boosting the DNA-primed immune response with recombinantMVA. In a second experiment using ten mice per group the enhancedimmunogenicity of the DNA/MVA sequence was confirmed: DNA/MVA 856±201;MVA/DNA 168±72; MVA/MVA 345±90; DNA/DNA 92±46. Therefore the sequence ofa first immunisation with a recombinant plasmid encoding the CS proteinfollowed by a second immunisation with the recombinant MVA virus yieldedthe highest levels of CD8+ T lymphocyte response after immunisation.

FIG. 3 shows malaria CD8 T cell ELISPOT data following differentimmunisation regimes. Results are shown as the number ofpeptide-specific T cells per million splenocytes. Mice were immunisedeither with the PbCSP-plasmid DNA or the PbCSP-MVA virus or combinationsof the two as shown on the X axis, at two week intervals and the numberof splenocytes specific for the pb9 malaria epitope assayed two weeksafter the last immunisation. Each point represents the number ofspot-forming cells (SFCs) measured in an individual mouse. The highestlevel of CD8+ T cells was induced by priming with the plasmid DNA andboosting with the recombinant MVA virus. This was more immunogenic thanthe reverse order of immunisation (MVA/DNA), two DNA immunisations(DNA/DNA) or two MVA immunisations (MVA/MVA). It was also moreimmunogenic than the DNA and MVA immunisations given simultaneously(DNA+MVA 2w), than one DNA immunisation (DNA 4w) or one MVA immunisationgiven at the earlier or later time point (MVA 2w and MVA 4w).

FIGS. 4A-4D shows that malaria CD8 T cell ELISPOT and CTL levels aresubstantially boosted by a recombinant MVA immunisation followingpriming with a plasmid DNA encoding the same antigen. A AND C. CD8+ Tcell responses were measured in BALB/c mice using the g-interferonELISPOT assay on fresh splenocytes incubated for 18 h with the K^(d)restricted peptide SYIPSAEKI [SEQ ID NO: 67] from P. berghei CSP and theL^(d) restricted peptide TPHPARIGL [SEQ ID NO: 69] from E. coliÿ-galactosidase. Note that the ELISPOT counts are presented on alogarithmic scale. B and D. Splenocytes from the same mice were alsoassayed in conventional ⁵¹Cr-release assays at an effector: targetration of 100:1 after 6 days of in vitro restimulation with the samepeptides (1 ÿg/ml).

The mice were immunised with plasmid DNA expressing either P. bergheiCSP and TRAP, PbCSP alone, the malaria epitope cassette including the P.berghei CTL epitope (labelled pTH.M), or ÿ-galactosidase. ELISPOT andCTL levels measured in mice 23 days after one DNA immunisation are shownin A and B respectively. The same assays were performed with animalsthat received additionally 1×10⁷ ffu of recombinant MVA expressing thesame antigen(s) two weeks after the primary immunisation. The ELISPOTand CTL levels in these animals are shown in C and D respectively. Eachbar represents data from an individual animal.

Studies were also undertaken of the immunogenicity of the epitope stringHM comprising both HIV and malaria epitopes in tandem. Using thisepitope string again the highest levels of CD8+ T cells and CTL weregenerated in the spleen when using an immunisation with DNA vaccinefollowed by an immunisation with a recombinant MVA vaccine (Table 4,FIG. 5).

TABLE 4 Immunogenicity of Various DNA/MVA Combinations as Determined byElispot Assays Immunisation 1 Immunisation 2 HIV epitope Malaria epitopeDNA-HM DNA-HM 56 ± 26 4 ± 4 MVA-HM MVA-HM 786 ± 334 238 ± 106 MVA-HMDNA-HM 306 ± 78  58 ± 18 DNA-HM MVA-HM 1000 ± 487  748 ± 446 None DNA-HM70 ± 60 100 ± 10  None MVA-HM 422 ± 128 212 ± 94 

Table 4 shows the results of ELISPOT assays performed to measure thelevels of specific CD8+ T cells to HIV and malaria epitopes followingdifferent immunisation regimes of plasmid DNA and MVA as indicated. Thenumbers are spot-forming cells per million splenocytes. The HM epitopestring is illustrated in FIG. 2. BALB/c mice were used in all cases. Themalaria epitope was pb9 as in FIGS. 2 and 3. The HIV epitope wasRGPGRAFVTI [SEQ ID NO: 51]. The immunisation doses were 50 ÿg of plasmidDNA or 10⁷ focus-forming units (ffu) of recombinant MVA. Allimmunisations were intramuscular. The interval between immunisations 1and 2 was from 14-21 days in all cases.

FIG. 5 shows the CTL responses induced in BALB/c mice to malaria and HIVepitopes by various immunisation regimes employing plasmid DNA andrecombinant MVA. Mice were immunised intramuscularly as described in thelegend to table 3 and in methods. High levels of CTL (>30% specificlysis at effector/target ration of 25:1) were observed to both themalaria and HIV epitopes only after priming with plasmid DNA andboosting with the recombinant MVA. The antigen used in this experimentis the HIV-malaria epitope string. The recombinant MVA is denoted MVA.HMand the plasmid DNA expressing this epitope string is denoted pTH.HM.Levels of specific lysis at various effector to target ratios are shown.These were determined after 5 days in vitro restimulation of splenocyteswith the two peptides pb9 and RGPGRAFVTI [SEQ ID NO: 51].

Comparison of numerous delivery systems for the induction of CTL wasreported by Allsopp et al. (1996). Recombinant Ty-virus like particles(Ty-VLPs) and lipid-tailed malaria peptides both gave good CTL inductionbut Ty-VLPs were better in that they required only a single immunisingdose for good CTL induction. However, as shown here even two doses of Typarticles fail to induce significant protection against sporozoitechallenge (Table 7, line 1). Immunisation with a recombinant modifiedvaccinia Ankara virus encoding the circumsporozoite protein of P.berghei also generates good levels of CTL. However, a much higher levelof CD8+ T cell response is achieved by a first immunisation with theTy-VLP followed by a second immunisation with the MVA CS vaccine (Table5).

TABLE 5 Immunogenicity of Various Ty-VLP/MVA Combinations as Determinedby ELISPOT and CTL Assays Immunisation 1 Immunisation 2 ELISPOT No %Specific Lysis Ty-CABDH Ty-CABDH 75 15 MVA.PbCSP MVA.PbCSP 38 35Ty-CABDH MVA.PbCSP 225 42 Ty-CABDH MVA.HM 1930 nd

Table 5 Results of ELISPOT and CTL assays performed to measure thelevels of specific CD8+ T cells to the malaria epitope pb9 followingdifferent immunisation regimes of Ty-VLPs and recombinant MVA virus asindicated. The CTL and ELISPOT data are from different experiments. TheELISPOT levels (spots per million splenocytes) are measured onun-restimulated cells and the CTL activity, indicated as specific lysisat an effector to target ratio of 40:1, on cells restimulated with pb9peptide in vitro for 5-7 days. Both represent mean levels of three mice.BALB/c mice were used in all cases. The immunisation doses were 50 ÿg ofTy-VLP or 10⁷ ffu (foci forming units) of recombinant MVA. Allimmunisations were intramuscular. The interval between immunisations 1and 2 was from 14-21 days. MVA.HM includes cassettes CAB.

Priming of an Immune Response with DNA Delivered by a Gene Gun andBoosting with Recombinant MVA

Immunogenicity and Challenge

The use of a gene gun to deliver plasmid DNA intradermally and therebyprime an immune response that could be boosted with recombinant MVA wasinvestigated. Groups of BALB/c mice were immunised with the followingregimen:

-   -   I) Three gene gun immunisations with pTH.PbCSP (4 mg per        immunisation) at two week intervals    -   II) Two gene gun immunisations followed by MVA i.v. two weeks        later    -   III) One intramuscular DNA immunisation followed by MVA i.v. two        weeks later.

The immunogenicity of the three immunisation regimens was analysed usingELISPOT assays. The highest frequency of specific T cells was observedwith two gene gun immunisations followed by an MVA i.v. boost and theintramuscular DNA injection followed an MVA i.v. boost (FIG. 6).

FIG. 6 shows the results of ELISPOT assays performed to measure thelevels of specific CD8+ T cells to the malaria epitope pb9 followingdifferent immunisation regimes. Groups of BALB/c mice (n=3) wereimmunised as indicated (g.g.=gene gun). The time between allimmunisations was 14 days. ELISPOT assays were done two weeks after thelast immunisation.

CTL Induction to the Same Antigen in Different Mouse Strains

To address the question whether the boosting effect described above inBALB/c mice with two CTL epitopes SYIPSAEKI [SEQ ID NO: 67] derived fromP. berghei CSP and RGPGRAFVTI [SEQ ID NO: 68] derived from HIV is auniversal phenomenon, two sets of experiments were carried out. CTLresponses to the influenza nucleoprotein were studied in five inbredmouse strains. In a first experiment three published murine CTL epitopesderived from the influenza nucleoprotein were studied (see Table 3).Mice of three different H-2 haplotypes, BALB/c and DBA/2 (H-2^(d)),C57BL/6 and 129 (H-2^(b)); CBA/J (H-2^(k)), were used. One set ofanimals was immunised twice at two week intervals with the plasmidV1J-NP encoding the influenza nucleoprotein. Another set of identicalanimals was primed with V1J-NP and two weeks later boosted intravenouslywith 10⁶ ffu of MVA.NP, expressing influenza virus NP. The levels of CTLin individual mice were determined in a ⁵¹Cr-release assay with peptidere-stimulated splenocytes. As shown in FIGS. 7A-7E, the DNA priming/MVAboosting immunisation regimen induced higher levels of lysis in all themouse strains analysed and is superior to two DNA injections.

FIGS. 7A-7E show the CTL responses against influenza NP in differentmouse strains. Mice of different strains were immunised twice two weeksapart with a DNA vaccine V1J-NP encoding for the influenza nucleoprotein(open circles) or primed with the same DNA vaccine and two weeks laterboosted with recombinant MVA expressing influenza virus nucleoprotein(closed circles). Two weeks after the last immunisation splenocytes wererestimulated in vitro with the respective peptides (Table 3). The CTLactivity was determined in a standard ⁵¹Cr-release assay with MHC classI-matched target cells.

CTL Induction to Different Antigens in Different Mouse Strains

The effect of MVA boosting on plasmid DNA-primed immune responses wasfurther investigated using different antigens and different inbred mousestrains. Mice of different strains were immunised with differentantigens using two DNA immunisations and compared with DNA/MVAimmunisations. The antigens used were E. coli ÿ-galactosidase, themalaria/HIV epitope string, a murine tumour epitope string and P.falciparum TRAP. Compared with two DNA immunisations theDNA-priming/MVA-boosting regimen induced higher levels of CTL in all thedifferent mouse strains and antigen combinations tested (FIGS. 8A-8H).

FIGS. 8A-8H show CTL responses against different antigens induced indifferent inbred mouse strains. Mice were immunised with two DNA vaccineimmunisations two weeks apart (open circles) or primed with a DNAvaccine and two weeks later boosted with a recombinant MVA expressingthe same antigen (closed circles). The strains and antigens were:C57BL/6; P. falciparum TRAP in A. DBA/2; E. coli b-galactosidase in B.BALB/c; HM epitope string CTL activity against malaria peptide (pb9) inC. DBA/2; HM epitope string CTL activity against pb9 in D. BALB/c; HMepitope string CTL activity against HIV peptide in E. DBA/2; HM epitopestring CTL activity against HIV peptide in F. BALB/c; tumour epitopestring CTL activity against P1A-derived peptide in G. DBA/2; tumourepitope string CTL activity against P1A-derived peptide in H. Sequencesof peptide epitopes are shown in table 3. Each curve shows the data foran individual mouse.

Sporozoites Can Efficiently Prime an Immune Response that is Boostableby MVA

Humans living in malaria endemic areas are continuously exposed tosporozoite inoculations. Malaria-specific CTL are found in thesenaturally exposed individuals at low levels. To address the questionwhether low levels of sporozoite induced CTL responses can be boosted byMVA, BALB/c mice were immunised with irradiated (to prevent malariainfection) P. berghei sporozoites and boosted with MVA. Two weeks afterthe last immunisation splenocytes were re-stimulated and tested forlytic activity. Two injections with 50 or 300+500 sporozoites inducedvery low or undetectable levels of lysis. Boosting with MVA induced highlevels of peptide specific CTL. MVA alone induced only moderate levelsof lysis (FIGS. 9A-9E).

FIGS. 9A-9E show sporozoite-primed CTL responses are substantiallyboosted by MVA. Mice were immunised with two low doses (50+50) ofirradiated sporozoites in FIG. 9A; two high doses (300+500) ofsporozoites in FIG. 9B; mice were boosted with MVA.PbCSP followinglow-dose sporozoite priming in FIG. 9D; high dose sporozoite priming inFIG. 9E. CTL responses following immunisation with MVA.PbCSP are shownin FIG. 9C.

Recombinant Adenoviruses as Priming Agent

The prime-boost immunisation regimen has been exemplified using plasmidDNA and recombinant Ty-VLP as priming agent. Here an example usingnon-replicating adenoviruses as the priming agent is provided.Replication-deficient recombinant Adenovirus expressing E. coliÿ-galactosidase (Adeno-GAL) was used. Groups of BALB/c mice wereimmunised with plasmid DNA followed by MVA or with Adenovirus followedby MVA. All antigen delivery systems used encoded E. coliÿ-galactosidase. Priming a CTL response with plasmid DNA or Adenovirusand boosting with MVA induces similar levels of CTL (FIGS. 10A-10B).

FIGS. 10A-10B show CTL responses primed by plasmid DNA or recombinantAdenovirus and boosted with MVA. Groups of BALB/c mice (n=3) were primedwith plasmid DNA (FIG. 10A); or recombinant Adenovirus expressingÿ-galactosidase (FIG. 10B). Plasmid DNA was administeredintramuscularly, MVA intravenously and Adenovirus intradermally.Splenocytes were restimulated with peptide TPHPARIGL [SEQ ID NO: 69] twoweeks after the last immunisation. CTL activity was tested withpeptide-pulsed P815 cells.

Immunogenicity of the DNA Prime Vaccinia Boost Regimen Depends on theReplication Competence of the Strain of Vaccinia Virus Used

The prime boosting strategy was tested using different strains ofrecombinant vaccina viruses to determine whether the different strainswith strains differing in their replication competence may differ intheir ability to boost a DNA-primed CTL response. Boosting withreplication-defective recombinant vaccinia viruses such as MVA and NYVACresulted in the induction of stronger CTL responses compared to CTLresponses following boosting with the same dose of replication competentWR vaccinia virus (FIGS. 11A-11C).

FIGS. 11A-11C show CTL responses in BALB/c mice primed with plasmid DNAfollowed by boosting with different recombinant vaccinia viruses.Animals were primed with pTH.PbCSP 50 ÿg/mouse i.m. and two weeks laterboosted with different strains of recombinant vaccina viruses (10⁶ pfuper mouse i.v.) expressing PbCSP. The different recombinant vacciniavirus strains were MVA in FIG. 11A; NYVAC in FIG. 11B and WR in FIG.11C. The superiority of replication-impaired vaccinia strains overreplicating strains was found in a further experiment. Groups of BALB/cmice (n=6) were primed with 50 ÿg/animal of pSG2.PbCSP (i.m.) and 10days later boosted i.v. with 10⁶ ffu/pfu of recombinant MVA, NYVAC andWR expressing PbCSP. The frequencies of peptide-specific CD8+ T cellswere determined using the ELISPOT assay. The frequencies were: MVA1103±438, NYVAC 826±249 and WR 468±135. Thus using both CTL assays andELISPOT assays as a measure of CD8 T cell immunogenicity a surprisingsubstantially greater immunogenicity of the replication-impairedvaccinia strains was observed compared to the replication competentstrain.

The Use of Recombinant Canary or Fowl Pox Viruses for Boosting Cd8+ TCell Responses

Recombinant canary pox virus (rCPV) or fowl pox virus (rFVP) are madeusing shuttle vectors described previously (Taylor et al. Virology 1992,187: 321-328 and Taylor et al. Vaccine 1988, 6: 504-508). The strategyfor these shuttle vectors is to insert the gene encoding the protein ofinterest preceded by a vaccinia-specific promoter between two flankingregions comprised of sequences derived from the CPV or FPV genome. Theseflanking sequences are chosen to avoid insertion into essential viralgenes. Recombinant CPV or FPV are generated by in vivo recombination inpermissive avian cell lines i.e. primary chicken embryo fibroblasts. Anyprotein sequence of antigens or epitope strings can be expressed usingfowl pox or canary pox virus. Recombinant CPV or FPV is characterisedfor expression of the protein of interest using antigen-specificantibodies or including an antibody epitope into the recombinant gene.Recombinant viruses are grown on primary CEF. An immune response isprimed using plasmid DNA as described in Materials and Methods. Thisplasmid DNA primed immune response is boosted using 10⁷ ffu/pfu of rCPVor rFPV inoculated intravenously, intradermally or intramuscularly. CD8+T cell responses are monitored and challenges are performed as describedherein.

Example 3 Malaria Challenge Studies in Mice

To assess the protective efficacy of the induced levels of CD8+ T cellresponse immunised BALB/c or C57BL/6 mice were challenged by intravenousinjection with 2000 or 200 P. berghei sporozoites. This leads toinfection of liver cells by the sporozoites. However, in the presence ofa sufficiently strong T lymphocyte response against the intrahepaticparasite no viable parasite will leave the liver and no blood-stageparasites will be detectable. Blood films from challenged mice weretherefore assessed for parasites by microscopy 5-12 days followingchallenge.

BALB/c mice immunised twice with a mixture of two plasmid DNAs encodingthe CS protein and the TRAP antigen, respectively, of P. berghei werenot protected against sporozoite challenge. Mice immunised twice with amixture of recombinant MVA viruses encoding the same two antigens werenot protected against sporozoite challenge. Mice immunised first withthe two recombinant MVAs and secondly with the two recombinant plasmidswere also not protected against sporozoite challenge. However, all 15mice immunised first with the two plasmid DNAs and secondly with the tworecombinant MVA viruses were completely resistant to sporozoitechallenge (Table 6 A and B).

To assess whether the observed protection was due to an immune responseto the CS antigen or to TRAP or to both, groups of mice were thenimmunised with each antigen separately (Table 6 B). All 10 miceimmunised first with the CS plasmid DNA and secondly with the CS MVAvirus were completely protected against sporozoite challenge. Fourteenout of 16 mice immunised first with the TRAP plasmid DNA vaccine andsecondly with the TRAP MVA virus were protected against sporozoitechallenge. Therefore the CS antigen alone is fully protective when theabove immunisation regime is employed and the TRAP antigen issubstantially protective with the same regime.

The good correlation between the induced level of CD8+ T lymphocyteresponse and the degree of protection observed strongly suggests thatthe CD8+ response is responsible for the observed protection. Inprevious adoptive transfer experiments it has been demonstrated thatCD8+ T lymphocyte clones against the major CD8+ T cell epitope in the P.berghei CS protein can protect against sporozoite challenge. Todetermine whether the induced protection was indeed mediated by CD8+ Tcells to this epitope we then employed a plasmid DNA and a recombinantMVA encoding only this nine amino acid sequence from P. berghei as apart of a string of epitopes (Table 6 B). (All the other epitopes werefrom micro-organisms other than P. berghei). Immunisation of 10 micefirst with a plasmid encoding such an epitope string and secondly with arecombinant MVA also encoding an epitope string with the P. berghei CTLepitope led to complete protection from sporozoite challenge (Table 6B). Hence the induced protective immune response must be the CTLresponse that targets this nonamer peptide sequence.

TABLE 6 Results of Mouse Challenge Experiments Using DifferentCombinations of DNA and MVA Vaccine No. Infected/ Immunisation 1Immunisation 2 No. challenged % Protection A. Antigens used: PbCSP +PbTRAP DNA DNA 5/5 0% MVA MVA  9/10 10% DNA MVA 0/5 100% MVA DNA 5/5 0%Control mice immunised with ÿ-galactosidase DNA DNA 5/5 0% MVA MVA 5/50% DNA MVA 5/5 0% MVA DNA 5/5 0% B. DNA(CSP + MVA (CSP + TRAP)  0/10100% TRAP) DNA (CSP) MVA (CSP)  0/10 100% DNA (TRAP) MVA (TRAP)  2/1688% DNA (epitope) MVA (epitope)  0/11 100% DNA (beta-gal) MVA (beta-gal)6/7 14% none none  9/10 10%

Table 6 Results of Two Challenge Experiments (A and B) Using DifferentImmunisation regimes of plasmid DNA and MVA as indicated. BALB/c micewere used in all cases. The immunisation doses were 50 ÿg of plasmid DNAor 10⁶ ffu of recombinant MVA. The interval between immunisations 1 and2 was from 14-21 days in all cases. Challenges were performed at 18-29days after the last immunisation by i.v. injection of 2000 P. bergheisporozoites and blood films assessed at 5, 8 and 10 days post challenge.CSP and TRAP indicate the entire P. berghei antigen and ‘epitope’indicates the cassettes of epitopes shown in table 1 containing only asingle P. berghei K^(d−) restricted nonamer CTL epitope. Note that inexperiment B immunisation with the epitope string alone yields 100%protection.

Mice immunised twice with recombinant Ty-VLPs encoding pb9 were fullysusceptible to infection. Similarly mice immunised twice with therecombinant MVA encoding the full CS protein were fully susceptible toinfection. However, the mice immunised once with the Ty-VLP andsubsequently once with the recombinant MVA showed an 85% reduction inmalaria incidence when boosted with MVA expressing the full length CSprotein, and 95% when MVA expressing the HM epitope string whichincludes pb9 was used to boost (Table 7).

TABLE 7 Results of Challenge Experiments Using Different ImmunisationRegimes of Ty-VLPs and MVA No. Infected/ Immunisation 1 Immunisation 2No. challenged % Protection Ty-CABDHFE Ty-CABDHFE 7/8 13% Ty-CABDHMVA.PbCSP  2/13 85% Ty-CABDHFE MVA-NP 5/5 0% MVA.PbCSP MVA.PbCSP 6/6 0%MVA.HM Ty-CABDHFE 14/14 0% Ty-CABDHFE MVA.HM  1/21 95% none MVA.HM 8/80% none none 11/12 9%

Table 7 Results of Challenge Experiments Using Different ImmunisationRegimes of Ty-VLPs and MVA as Indicated. BALb/c Mice Were Used in AllCases.

Immunisations were of 50 ÿg of Ty-VLP or 10 ffu of recombinant MVAadministered intravenously. The interval between immunisations 1 and 2was from 14-21 days in all cases. Challenges were performed at 18-29days after the last immunisation by i.v. injection of 2000 P. bergheisporozoites and blood films assessed at 5, 8 and 10 days post challenge.CSP indicates the entire P. berghei antigen. Ty-VLPs carried epitopecassettes CABDH or CABDHFE as described in table 1. MVA.HM includescassettes CAB.

To determine whether the enhanced immunogenicity and protective efficacyobserved by boosting with a recombinant MVA is unique to this particularvaccinia virus strain or is shared by other recombinant vaccinias thefollowing experiment was performed. Mice were immunised with the DNAvaccine encoding P. berghei CS protein and boosted with either (i)recombinant MVA encoding this antigen; (ii) recombinant wild-typevaccinia virus (Western Reserve strain) encoding the same antigen(Satchidanandam et al. 1991), or (iii) recombinant NYVAC (COPAK) virus(Lanar et al. 1996) encoding the same malaria antigen. The highestdegree of protection was observed with boosting by the MVA recombinant,80% (Table 8). A very low level of protection (10%) was observed byboosting with the wild-type recombinant vaccinia virus and a significantlevel of protection, 60%, by boosting with the NYVAC recombinant. Hencethe prime-boost regime we describe induces protective efficacy with anynon-replicating vaccinia virus strain. Both the MVA recombinant andNYVAC were significantly (P<0.05 for each) better than the WR strainrecombinant.

TABLE 8 Challenge Data Results for DNA Boosted with Various VacciniaStrain Recombinants. No. Infected/ Immunisation 1 Immunisation 2 No.challenged % Protection DNA-beta gal. MVA.NP 8/8  0% DNA-CSP MVA-CSP2/10 80% DNA-CSP WR-CSP 9/10 10% DNA-CSP NYVAC-CSP 4/10 60%

Table 8 Results of a challenge experiment using different immunisationregimes of plasmid DNA and various vaccinia recombinants as indicated.BALB/c mice were used in all cases. The immunisation doses were 50 ÿ gof plasmid DNA or 10⁶ ffu/pfu of recombinant MVA or 10⁴ ffu/pfu ofrecombinant wild type (WR) vaccinia or 10⁶ ffu/pfu of recombinant NYVAC.Because the WR strain will replicate in the host and the other strainswill not, in this experiment a lower dose of WR was used. The intervalbetween immunisations 1 and 2 was 23 days. Challenges were performed at28 days after the last immunisation by i.v. injection of 2000 P. bergheisporozoites and blood films assessed at 7, 9 and 11 days post challenge.pbCSP indicates the entire P. berghei antigen and NP the nucleoproteinantigen of influenza virus (used as a control antigen). The firstimmunisation of group A mice was with the plasmid DNA vector expressingbeta galactosidase but no malaria antigen.

In a further experiment shown in Table 8, mice were immunised with theDNA vaccine encoding P. berghei CS protein and boosted with either (i)recombinant MVA encoding this antigen; (ii) recombinant WR vacciniavirus encoding the same antigen or (iii) recombinant NYVAC (COPAK) virusencoding the same malaria antigen, all at 10⁶ ffu/pfu. A high andstatistically significant degree of protection was observed withboosting with recombinant NYVAC (80%) or recombinant MVA (66%). A lowand non-significant level of protection (26%) was observed by boostingwith the WR recombinant vaccinia virus (Table 9). MVA and NYVAC boostingeach gave significantly more protection than WR boosting (P=0.03 andP=0.001 respectively). These data reemphasise that non-replicating poxvirus strains are better boosting agents for inducing high levels ofprotection.

TABLE 9 Influence of Different Recombinant Vaccinia Strains onProtection. Immunisation 1 No. inf./ DNA Immunisation 2 No. chall. %protection CSP MVA.PbCSP  5/15 66 CSP NYVAC.PbCSP  2/15 80 CSP WR.PbCSP11/15 26 ÿ-galactosidase MVA.NP 8/8 0

Table 9 Results of challenge experiments using different immunisationregimes of plasmid DNA and replication incompetent vaccinia recombinantsas boosting immunisation. BALB/c mice were used in all cases. Theimmunisation doses were 50 ÿg of plasmid DNA or 10⁶ ffu/pfu ofrecombinant MVA or recombinant wild type (WR) vaccinia or recombinantNYVAC. The interval between immunisations 1 and 2 was 23 days.Challenges were performed at 28 days after the last immunisation by i.v.injection of 2000 P. berghei sporozoites and blood films assessed at 7,9 and 11 days post challenge. PbCSP indicates the entire P. bergheiantigen and NP the nucleoprotein antigen of influenza virus (used as acontrol antigen). The control immunisation was with a plasmid DNA vectorexpressing ÿ-galactosidase followed by MVA.NP.

Alternative Routes for Boosting Immune Responses with Recombinant MVA

Intravenous injection of recombinant MVA is not a preferred route forimmunising humans and not feasible in mass immunisations. Thereforedifferent routes of MVA boosting were tested for their immunogenicityand protective efficacy.

Mice were primed with plasmid DNA i.m. Two weeks later they were boostedwith MVA administered via the following routes: intravenous (i.v.),subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.p.) andintradermal (i.d.). Two weeks after this boost peptide-specific CD8+ Tcells were determined in an ELISPOT assay. The most effective routewhich induced the highest levels were i.v. and i.d inoculation of MVA.The other routes gave moderate to poor responses (FIG. 12).

FIG. 12 shows frequencies of peptide-specific CD8+ T cells followingdifferent routes of MVA boosting. Results are shown as the number ofspot-forming cells (SFC) per one million splenocytes. Mice were primedwith plasmid DNA and two weeks later boosted with MVA via the indicatedroutes. The number of splenocytes specific for the SYIPSAEKI [SEQ ID NO:67] peptide was determined in INF-ÿ ELISPOT assays two weeks after thelast immunisation. Each bar represents the mean number of SFCs fromthree mice assayed individually.

Boosting via the i.v. route was compared with the i.d. and i.m route ina challenge experiment. The i.d route gave high levels of protection(80% protection). In the group of animals that were boosted via the i.m.route, 50% of the animals were protected. Complete protection wasachieved with MVA boost administered i.v. (Table 10)

TABLE 10 Influence of the Route of MVA Administration on ProtectiveEfficacy No. infected/ Immunisation 1 Immunisation 2 No. DNA MVAchallenged % protection CSP CSP i.v. *0/20  100 CSP CSP i.d 2/10 80 CSPCSP i.m. 5/10 50 Epitope epitope i.v. 1/10 90 NP NP i.v. 10/10  0*culminative data from two independent experiments

Table 10 Results from challenge experiments using different routes ofMVA boosting immunisation. Animals were primed by intramuscular plasmidDNA injection and two weeks later boosted with the indicated recombinantMVA (10⁶ ffu/mouse) administered via the routes indicated. The mice werechallenged 16 days after the last immunisation with 2000 P. bergheisporozoites and screened for blood stage parasitemia at day 8 and 10post challenge. Epitope indicates the polypeptide string HM.

Alternative Routes of DNA Priming: The Use of a Gene Gun to PrimePeptide Specific Cd8+ T Cells

Gene gun delivery is described in detail in for example in Eisenbraun etal. DNA Cell Biol. 1993, 12: 791-797 and Degano et al. Vaccine 1998, 16:394-398.

The mouse malaria challenge experiments described so far using plasmidDNA to prime an immune response used intramuscular injection of plasmidDNA.

Intradermal delivery of plasmid DNA using a biolistic device is anotherroute to prime specific CTL responses. Plasmid DNA is coated onto goldparticles and delivered intradermally with a gene gun. Groups of mice(n=10) were immunised three times at two weeks intervals with the genegun alone (4 ÿg/immunisation), immunised two times with the gene gunfollowed by an intravenous MVA.PbCSP boost or immunised intramuscularlywith 50 ÿg of pTH.PbCSP and two weeks later boosted with MVA.PbCSPintravenously. Two weeks after the last immunisation the animals werechallenged with 2000 sporozoites to assess protective efficacy of eachimmunisation regimen. In the group that received the intravenous MVAboost following two gene gun immunisations one out of ten animalsdeveloped blood stage parasitemia (90% protection). Complete protectionwas observed with intramuscular DNA priming followed by MVA i.vboosting. Seven out of 10 animals that were immunised three times withthe gene gun were infected. (30% protection) (Table 11).

Immunisation 1 No. inf./ % DNA Immunisation 2 Immunisation 3 No. chall.protection gene gun DNA gene gun DNA gene gun DNA 7/10 30 gene gun DNAgene gun DNA MVA.PbCSP 1/10 90 — DNA i.m MVA.PbCSP 0/10 100 Naïve 10/10 0

Table 11 Results of challenge experiments comparing different routes ofDNA priming (intradermally by gene gun versus intramuscular needleinjection). Groups of BALB/c mice (n=10) were immunised as indicated.Each gene gun immunisation delivered 4 ÿg of plasmid DNAintraepidermally. For i.m. immunisations 50 mg of plasmid DNA wereinjected. Twenty days after the last immunisation mice were challengedas described previously.

Highly Susceptible C57BL/6 Mice Are Protected

C57BL/6 mice are very susceptible to P. berghei sporozoite challenge.C57BL/6 mice were immunised using the DNA-MVA prime boost regime withboth pre-erythrocytic antigens PbCSP and PbTRAP, and challenged witheither 200 or 1000 infectious sporozoites per mouse. (Two hundredsporozoites corresponds to more than twice the dose required to induceinfection in this strain). All ten mice challenged with 200 sporozoitesshowed sterile immunity. Even the group challenged with 1000sporozoites, 60% of the mice were protected (Table 12). All the naiveC57BL/6 mice were infected after challenge.

TABLE 12 Protection of C57BL/6 Mice from Sporozoite Challenge No.animals inf./ % No. challenged protection 1000 sporozoites DNA followedby MVA  4/10 60 Naïve 5/5 0 200 sporozoites Naïve 5/5 0

Table 12 Results of a challenge experiment using C57BL/6 mice. Animalswere immunised with PbCSP and PbTRAP using the DNA followed by MVA primeboost regime. Fourteen days later the mice were challenged with P.berghei sporozoites as indicated.

Example 4

Protective Efficacy of the DNA-Priming/MVA-Boosting Regimen in TwoFurther Disease Models in Mice

Following immunogenicity studies, the protective efficacy of theDNA-priming MVA-boosting regimen was tested in two additional murinechallenge models. The two challenge models were the P815 tumour modeland the influenza A virus challenge model. In both model systems CTLhave been shown to mediate protection.

P815 Tumour Challenges:

Groups (n=10) of DBA/2 mice were immunised with a combination of DNAfollowed by MVA expressing a tumour epitope string or the HM epitopestring. Two weeks after the last immunisation the mice were challengedintravenously with 10⁵ P815 cells. Following this challenge the micewere monitored regularly for the development of tumour-related signs andsurvival.

FIG. 13 shows the survival rate of the two groups of mice. Sixty daysafter challenge eight out of ten mice were alive in the group immunisedwith the tumour epitopes string. In the group immunised with the HMepitope string only 2 animals survived. This result is statisticallysignificant: 2/10 vs 8/10 chi-squared=7.2. P=0.007. The onset of deathin the groups of animals immunised with the tumour epitope string isdelayed compared to the groups immunised with the HM epitope string.

Influenza Virus Challenges:

Groups of BALB/c mice were immunised with three gene gun immunisationswith plasmid DNA, two intramuscular plasmid DNA injections, one i.m. DNAinjection followed by one MVA.NP boost i.v. or two gene gunimmunisations followed by one MVA.NP boost i.v. Plasmid DNA andrecombinant MVA expressed the influenza virus nucleoprotein. Two weeksafter the last immunisation the mice were challenged intranasally with100 HA of influenza A/PR/8/34 virus. The animals were monitored forsurvival daily after challenge.

Complete protection was observed in the following groups of animals:

-   -   two DNA gene gun immunisations followed by one MVA.NP boost        i.v.;    -   one i.m. DNA injection followed by one MVA.NP boost i.v.; and    -   two i.m. DNA injections.

In the group of animals immunised three times with the gene gun 71% ofthe animals survived (5/7) and this difference from the control groupwas not significant statistically (P>0.05). In the naive group 25% ofthe animals survived (FIG. 14) and this group differed significantly(P<0.05) for the two completely protected groups.

FIG. 14 shows results of an influenza virus challenge experiment. BALB/cmice were immunised as indicated. GG=gene gun immunisations,im=intramuscular injection, iv=intravenous injection. Survival of theanimals was monitored daily after challenge. In a second experimentgroups of 10 BALB/c mice were immunised with MVA.NP i.v. alone, threetimes with the gene gun, two times with the gene gun followed by oneMVA.NP boost i.v. and two i.m injections of V1J-NP followed by oneMVA.NP boost. Two weeks after the last immunisation the mice werechallenged with 100 HA units of influenza A/PR/8/34 virus.

Complete and statistically significant protection was observed in thefollowing groups of animals:

two gene gun immunisations followed by one MVA.NP boost; and

two i.m injections of V1J-NP followed by one MVA.NP boost.

In the group receiving one MVA.NP i.v., 30% (3 out of 10) of animalssurvived. In the group immunised with a DNA vaccine delivered by thegene gun three times, 70% of the animals were protected but thisprotection was not significantly different from the naïve controls. Inthis challenge experiment 40% (4 out of 10) of the naive animalssurvived the challenge.

Example 5 Immunogenicity Studies in Non-Human Primates Immunogenicityand Protective Efficacy of the Prime Boost Regimen in Non-HumanPrimates.

In order to show that the strong immunogenicity of the DNA priming/MVAboosting regime observed in mice translates into strong immunogenicityin primates, the regimen was tested in macaques. The vaccine consistedof a string of CTL epitopes derived from HIV and SIV sequences (FIG. 2),in plasmid DNA or MVA, denoted DNA.H and MVA.H respectively. The use ofdefined CTL epitopes in a polyepitope string allows testing for SIVspecific CTL in macaques. Due to the MHC class I restriction of theantigenic peptides, macaques were screened for their MHC class Ihaplotype and Mamu-A*01-positive animals were selected for theexperiments described.

Three animals (CYD, DI and DORIS) were immunised following thisimmunisation regimen:

week 0 DNA (8ÿg, i.d., gene gun) week 8 DNA (8ÿg, i.d., gene gun) week17 MVA (5 × 10⁸ pfu, i.d.) week 22 MVA (5 × 10⁸ pfu, i.d.)

Blood from each animal was drawn at weeks 0, 2, 5, 8, 10, 11, 17, 18,19, 21, 22, 23, 24 and 25 of the experiment. The animals were monitoredfor induction of CTL using two different methods. PBMC isolated fromeach bleed were re-stimulated in vitro with a peptide encoded in theepitope string and tested for their ability to recognise autologouspeptide-loaded target cells in a chromium release cytotoxicity assay.Additionally, freshly isolated PBMC were stained for antigen specificCD8+ T cells using tetramers.

Following two gene gun immunisations very low levels of CTL weredetected using tetramer staining (FIGS. 15A-15C). Two weeks after thefirst MVA boosting, all three animals developed peptide specific CTL asdetected by tetramer staining (FIGS. 15A-15C). This was reflected by thedetection of moderate CTL responses following in vitro restimulation(FIG. 16B, week 19). The second boost with MVA.H induced very highlevels of CD8+, antigen specific T cells (FIGS. 15A-15C) and also veryhigh levels of peptide specific cytotoxic T cells (FIG. 16C, week 23).

FIGS. 15A-15C show detection of SIV-specific MHC class I-restricted CD8+T cells using tetramers. Three Mamu-A*A01-positive macaques wereimmunised with plasmid DNA (gene gun) followed by MVA boosting asindicated. Frequencies of Mamu-A*A01/CD8 double-positive T cells wereidentified following FACS analysis. Each bar represents the percentageof CD8+ T cells specific for the Mamu-A*01/gag epitope at the indicatedtime point. One percent of CD8 T cells corresponds to about 5000/10⁶peripheral blood lymphocytes. Thus the levels of epitope-specific CD8 Tcells in the peripheral blood of these macaques are at least as high asthe levels obvserved in the spleens of immunised and protected mice inthe malaria studies.

FIGS. 16A-16C shows CTL induction in macaques following DNA/MVAimmunisation. PBMC from three different macaques (CYD, DI and DORIS)were isolated at week 18, 19 and 23 and were restimulated with peptideCTPYDINQM [SEQ ID NO: 54] in vitro. After two restimulations withpeptide CTPYDINQM [SEQ ID NO: 54] the cultures were tested for theirlytic activity on peptide-pulsed autologous target cells. Strong CTLactivity was observed.

Example 6 Immunogenicity and Challenge Studies in Chimpanzees

To show that a similar regime of initial immunisation with plasmid DNAand subsequent immunisation with recombinant MVA can be effectiveagainst Plasmodium falciparum malaria in higher primates an immunisationand challenge study was performed with two chimpanzees. Chimp H1received an initial immunisation with

500 ÿg of a plasmid expressing Plasmodium falciparum TRAP from the CMVpromoter without intron A, CMV-TRAP. Chimp H2 received the same dose ofCMV-LSA-1, which expresses the C-terminal portion of the LSA-1 gene ofP. falciparum. Both chimps received three more immunisations over thenext 2 months, but with three plasmids at each immunisation. H1 receivedCMV-TRAP as before, plus pTH-TRAP, which expresses TRAP using the CMVpromoter with intron A, leading to a higher expression level. H1 alsoreceived RSV-LSA-1, which expresses the C-terminal portion of LSA-1 fromthe RSV promoter. H2 received CMV-LSA-1, pTH-LSA-1 and RSV-TRAP at thesecond, third and fourth immunisations. The dose was always 500 ÿg ofeach plasmid.

It was subsequently discovered that the RSV plasmids did not express theantigens contained within them, so H1 was only immunised with plasmidsexpressing TRAP, and H2 with plasmids expressing LSA-1.

Between and following these DNA immunisations assays of cellular immuneresponses were performed at several time points, the last assay beingperformed at three months following the fourth DNA immunisation, but nomalaria-specific T cells were detectable in either ELISPOT assays or CTLassays for CD8+ T cells.

Both animals were subsequently immunised with three doses of 10⁸ ffu ofa recombinant MVA virus encoding the P. falciparum TRAP antigen over a 6week period. Just before and also following the third recombinant MVAimmunisation T cell responses to the TRAP antigen were detectable inboth chimpanzees using an ELISPOT assay to whole TRAP protein bound tolatex beads. This assay detects both CD4+ and CD8+ T cell responses.Specific CD8+ T responses were searched for with a series of short 8-11amino acid peptides in both immunised chimpanzees. Such analysis forCD8+ T cell responses indicated that CD8+ T cells were detectable onlyin the chimpanzee H1. The target epitope of these CD8+ T lymphocytes wasan 11 amino acid peptide from TRAP, tr57, of sequence KTASCGVWDEW [SEQID NO: 78]. These CD8+ T cells from H1 had lytic activity againstautologous target cells pulsed with the tr57 peptide and againstautologous target cells infected with the recombinant PfTRAP-MVA virus.A high precursor frequency of these specific CD8+ T cells of about 1 per500 lymphocytes was detected in the peripheral blood of this chimpanzeeH1 using an ELISPOT assay two months following the final MVAimmunisation. No specific CD8+ T cell response was clearly detected inthe chimpanzee H2, which was not primed with a plasmid DNA expressingTRAP.

Two months after the third PfTRAP-MVA immunisation challenge of H1 andH2 was performed with 20,000 sporozoites, a number that has previouslybeen found to yield reliably detectable blood stage infection inchimpanzees 7 days after challenge (Thomas et al. 1994 and unpublisheddata). The challenge was performed with the NF54 strain of Plasmodiumfalciparum. This is of importance because the TRAP sequence in theplasmid DNA and in the recombinant MVA is from the T9/96 strain of P.falciparum which has numerous amino acid differences to the NF54 TRAPallele (Robson et al. 1990). Thus, this sporozoite challenge wasperformed with a heterologous rather than homologous strain of parasite.In the chimpanzee H2 parasites were detectable in peripheral blood asexpected 7 days after sporozoite challenge using in vitro parasiteculture detection. However, in H1 the appearance of blood stageparasites in culture from the day 7 blood samples was delayed by threedays consistent with some immune protective effect against theliver-stage infection. In studies of previous candidate malaria vaccinesin humans a delay in the appearance of parasites in the peripheral bloodhas been estimated to correspond to a substantial reduction in parasitedensity in the liver (Davis et al. 1989). Thus the chimpanzee HI,immunised first with P. falciparum TRAP plasmid DNA and subsequentlywith the same antigen expressed by a recombinant MVA virus showed astrong CD8+ T lymphocyte response and evidence of some protection fromheterologous sporozoite challenge.

Discussion

These examples demonstrate a novel regime for immunisation againstmalaria which induces high levels of protective CD8+ T cells in rodentmodels of human malaria infection. Also demonstrated is an unprecedentedcomplete protection against sporozoite challenge using subunit vaccines(36 out of 36 mice protected in Table 6 using DNA priming and MVAboosting with the CS epitope containing vaccines). Induction ofprotective immune responses using the DNA priming/MVA boosting regimenwas demonstrated in two additional mouse models of viral infectioninfluenza A model and cancer (P815 tumour model). More importantly forvaccines for use in humans this immunisation regimen is also highlyimmunogenic for CD8+ T cells in primates. Strong SIV-gag-specific CTLwere induced in 3 out of 3 macaques with plasmid DNA and MVA expressingepitope strings. The levels induced are comparable to those found inSIV-infected animals. The data from the chimpanzee studies indicate thatthe same immunisation regime can induce a strong CD8+ T lymphocyteresponse against P. falciparum in higher primates with some evidence ofprotection against P. falciparum sporozoite challenge.

Ty-VLPs have previously been reported to induce good levels of CD8+ Tcell responses against the P. berghei rodent malaria (Allsopp et al.1995) but alone this construct is not protective. It has now been foundthat subsequent immunisation with recombinant MVA boosts the CD8+ T cellresponse very substantially and generates a high level of protection(Table 7).

Recombinant MVA viruses have not been assessed for efficacy as malariavaccines previously. Recombinant MVA alone was not significantlyprotective, nor was priming with recombinant MVA followed by a secondimmunisation with recombinant plasmid DNA. However, a secondimmunisation with the recombinant MVA following an initial immunisationwith either Ty-VLPs or plasmid DNA yielded impressive levels ofprotection. Non-recombinant MVA virus has been safely used to vaccinatethousands of human against smallpox and appears to have an excellentsafety profile. The molecular basis of the increased safety andimmunogenicity of this strain of vaccinia virus is being elucidated bydetailed molecular studies (Meyer et al. 1991; Sutter at al. 1994).

Plasmid DNA has previously been tested as a malaria vaccine for the P.yoelii rodent malaria. High levels of, but not complete, protection isseen in some strains but in other strains of mice little or noprotection was observed even after multiple immunisations (Doolan et al.1996). Although plasmid DNA has been proposed as a method ofimmunisation against P. falciparum, success has not previously beenachieved. The evidence provided here is the first evidence to show thatplasmid DNA may be used in an immunisation regime to induce protectiveimmunity against the human malaria parasite P. falciparum.

A similar regime of immunisation to the regime demonstrated herein canbe expected to induce useful protective immunity against P. falciparumin humans. It should be noted that five of the vaccine constructsemployed in these studies to induce protective immunity in rodents orchimpanzees contain P. falciparum sequences and could therefore be usedfor human immunisation against P. falciparum. These are: 1. The P.falciparum TRAP plasmid DNA vaccine. 2. The P. falciparum TRAPrecombinant MVA virus. 3. The Ty-VLP encoding an epitope string ofnumerous P falciparum epitopes, as well as the single P. berghei CTLepitope. 4. The plasmid DNA encoding the same epitope string as 3. 5.The recombinant MVA encoding the longer HM epitope string including manyof the malaria epitopes in 3 and 4. Similarly the plasmid DNAs and MVAencoding HIV epitopes for human class I molecules could be used ineither prophylactic or therapeutic immunisation against HIV infection.

These studies have provided clear evidence that a novel sequentialimmunisation regime employing a non-replicating or replication-impairedpox virus as a boost is capable of inducing a strong protective CD8+ Tcell response against the malaria parasite. The examples demonstrateclearly a surprising and substantial enhancement of CD8+ T cellresponses and protection compared to replicating strains of pox viruses.Because there is no reason to believe that the immunogenicity of CD8+ Tcell epitopes from the malaria parasite should differ substantially fromCD8+ T cell epitopes in other antigens it is expected that theimmunisation regime described herein will prove effective at generatingCD8+ T cell responses of value against other diseases. The critical stepin this immunisation regimen is the use of non-replicating orreplication-impaired recombinant poxviruses to boost a pre-existing CTLresponse. We have shown that CTL responses can be primed using differentantigen delivery systems such as a DNA vaccine i.d. and i.m, arecombinant Ty-VLP, a recombinant adenovirus and irradiated sporozoites.This is supported by the data presented on the generation of a CD8+ Tcell response against HIV, influenza virus and tumours. Amongst severalknown examples of other diseases against which a CD8+ T cell immuneresponse is important are the following: infection and disease caused bythe viruses HIV, herpes simplex, herpes zoster, hepatitis C, hepatitisB, influenza, Epstein-Barr virus, measles, dengue and HTLV-1; by thebacteria Mycobacterium tuberculosis and Listeria sp.; and by theprotozoan parasites Toxoplasma and Trypanosoma. Induction of protectiveCTL responses against influenza A virus has been demonstrated in FIG.14. Furthermore, the immunisation regime described herein is expected tobe of value in immunising against forms of cancer where CD8+ T cellresponses plays a protective role. The induction of protective CTLresponses using the DNA prime MVA boost regime against tumours is shownin FIG. 13. Specific examples in humans include melanoma, cancer of thebreast and cancer of the colon.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

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1. A method for generating a CD8⁺ T cell immune response in a humanagainst human immunodeficiency virus (HIV) comprising administering tosaid human at least one dose of each of the following: (i) a primingcomposition comprising or encoding one or more CD8⁺ T cell epitopes ofHIV; and (ii) a boosting composition comprising a recombinant poxvirusvector encoding at least one of said one or more CD8⁺ T cell epitopes ofHIV, wherein the recombinant poxvirus vector is non-replicating orreplication-impaired in the human; wherein if the priming composition in(i) is a viral vector, then it is derived from a different virus thanthe poxvirus vector in (ii), and wherein a CD8⁺ T cell immune responseagainst said at least one CD8⁺ T cell epitope of HIV is generated in thehuman.
 2. The method of claim 1 wherein the non-replicating orreplication-impaired recombinant poxvirus vector is a recombinantvaccinia virus.
 3. The method of claim 2 wherein the recombinantvaccinia virus is a recombinant MVA vector.
 4. The method of claim 1wherein the non-replicating or replication-impaired recombinant poxvirusvector is a recombinant avipox virus.
 5. The method of claim 4 whereinthe recombinant avipox virus is a recombinant fowlpox vector.
 6. Themethod of claim 4 wherein the recombinant avipox virus is a recombinantcanarypox vector.
 7. The method of claim 6 wherein the recombinantcanarypox vector is a recombinant ALVAC vector.
 8. The method of claim 1wherein the priming composition is a recombinant DNA plasmid.
 9. Themethod of claim 1 wherein the priming composition is a viral vector. 10.The method of claim 9 wherein the viral vector is a herpes viral vector.11. The method of claim 9 wherein the viral vector is a replicatingviral vector.
 12. The method of claim 9 wherein the viral vector is anon-replicating or replication-impaired viral vector.
 13. (canceled) 14.(canceled)
 15. The method of claim 1 wherein the CD8⁺ T cell epitopesare one or more epitope strings comprising an amino acid sequenceselected from the group consisting of: SEQ ID Nos: 42, 43, 45-49, 51-53and 55-64.
 16. A method for generating a CD8⁺ T cell immune response ina human against human immunodeficiency virus (HIV) comprisingadministering to said human at least one dose of each of the following:(i) a priming composition comprising a DNA plasmid encoding one or moreCD8⁺ T cell epitopes of HIV; and (ii) a boosting composition comprisinga recombinant vaccinia virus encoding at least one of said one or moreCD8⁺ T cell epitopes of HIV, wherein the recombinant vaccinia virus isnon-replicating or replication-impaired in the human, wherein a CD8⁺ Tcell immune response against said at least one CD8⁺ T cell epitope ofHIV is generated in the human.
 17. The method of claim 16 wherein thenon-replicating or replication-impaired recombinant vaccinia virus is arecombinant MVA vector. 18-21. (canceled)
 22. A method for generating aCD8⁺ T cell immune response in a human against human immunodeficiencyvirus (HIV) comprising administering to said human at least one dose ofeach of the following: (i) a priming composition comprising a DNAplasmid encoding one or more CD8⁺ T cell epitopes of HIV; and (ii) aboosting composition comprising a recombinant poxvirus vector encodingat least one of said one or more CD8⁺ T cell epitopes of HIV, whereinthe recombinant poxvirus vector is non-replicating orreplication-impaired in the human; wherein a CD8⁺ T cell immune responseagainst said at least one CD8⁺ T cell epitope of HIV is generated in thehuman.
 23. (canceled)
 24. (canceled)
 25. The method of claim 22 whereinthe CD8⁺ T cell epitopes are one or more epitope strings comprising anamino acid sequence selected from the group consisting of: SEQ ID Nos:42, 43, 45-49, 51-53 and 55-64.
 26. (canceled)
 27. (canceled)
 28. Themethod of claim 16 wherein the CD8⁺ T cell epitopes are one or moreepitope strings comprising an amino acid sequence selected from thegroup consisting of: SEQ ID Nos: 42, 43, 45-49, 51-53 and 55-64. 29-31.(canceled)
 32. The method of claim 1 wherein the CD8⁺ T cell immuneresponse assists in controlling HIV infection